MEMBRANE AND CELLULAR BIOCHEMISTRY

60.346

LAB MANUAL

2002

Lab manual is available as a pdf file on the website.

TABLE OF CONTENTS

Lab # CONTENTS PAGE

______________________________________________________________________________

EXPERIMENT SCHEDULE 3

GENERAL INSTRUCTIONS 4

LAB STANDARD OPERATIONS PROCEDURE 8

WHMIS 10

1 THE ISOLATION AND QUANTITATION OF UBIQUINONE 12

Saponification 14

Extraction and Evaporation 15

TLC 17

Scanning spectrophotometry 19

2 THE ISOLATION OF RED BLOOD CELL MEMBRANE PROTEINS 22

Preparation of Red Blood Cell Membranes 24

Protein Determination 25

Polyacrylamide gel electrophoresis 25

Reagents 34

APPENDIX

Pipetman Operation 35

Centrifuge Operation (floor model) 37

Sorvall RC2-B
Himac

Spectronic 20D Operation 38

Ultrospec 4000 spectophotometer Operation 39

SAMPLE LAB EXAM 40

 3

60.346 SCHEDULE 2002

Lab # Experiment Week # Date

______________________________________________________________________________
SECTION 1
1 The isolation and quantitation of ubiquinone
(1) Saponification 1 Sept 13
(2) TLC 2 Sept 20
(3) Scanning spectrophotometry 3 Sept 27
Problem lab Sept 27
Lab report due Oct 11

2 The isolation of red blood cell membrane proteins

(1) Isolation and Bradford protein determination 7 Oct 25
(2) Lowry protein determination and Polyacrylamide 8 Nov 1
gel electrophoresis
Lab report due Nov 15

3 Tutorial and Problem Lab

Introduction to problem lab (starts at 2:30 pm in room 204) 9 Nov 8
Tutoriala (starts at 2:45 pm) 9 Nov 8
Problem lab (starts at 3:45 pm - room 204 or room 201) 9 Nov 8
a
refer to tutorial schedule for locations

SECTION 2
1 The isolation and quantitation of ubiquinone
(1) Saponification 4 Oct 4
(2) TLC 5 Oct 11
(3) Scanning spectrophotometry 6 Oct 18
Problem lab Oct 18
Lab report due Nov 1

2 The isolation of red blood cell membrane proteins

(1) Isolation and Bradford protein determination 7 Oct 25
(Room 201 Buller)
(2) Lowry protein determination and 8 Nov 1
Polyacrylamide gel electrophoresis (Room 201 Buller)
Lab report due Nov 15

3 Tutorial and Problem Lab

Introduction to problem lab (starts at 2:30 pm in room 204) 9 Nov 8
Problem labb (starts at 2:45 pm - room 204) 9 Nov 8
Tutorialc (starts at 3:45 pm) 9 Nov 8
b
refer to tutorial schedule for locations
c
if you have not completed the problem lab, return to lab to
complete the problem lab after the tutorial

SECTIONS 1 & 2
4 Question Lab (both sections, not compulsory) 11 Nov 22
Come prepared with questions.

All labs are in room 204 Buller except where indicated in the above schedule.

LAB EXAM Nov 29

 4

GENERAL INSTRUCTIONS

Lab Instructor: Dr. L. Cameron Office: 414B

Demonstrators: Andrew Schurko Lab: 401
Ayush Kumar Lab: 304

Lab Location: 204 Buller Bldg. (on occasion 201 Buller for section 2 - check schedule)

WEBSITE: www.umanitoba.ca/faculties/science/microbiology/staff/cameron/

OR via University of Manitoba Microbiology Homepage:

https://www.umanitoba.ca/faculties/science/microbiology/course_notes.html
Information available at the website: changes/corrections, additional information, data, marks

REGULATIONS
1. Lab attendance is compulsory.
2. Students must wear a lab coat.
3. There is no smoking, drinking, or eating in the lab.
4. Students work in pairs.
5. Each student or group's bench area must be thoroughly cleaned and wiped with cleaner
before leaving the lab. Marks can be subtracted from final lab mark for poor lab conduct.

EVALUATION

1. The lab is worth 20% of the final course mark.

Lab 1 report, The Isolation and Quantitation of Ubiquinone............. 1.5 %
Lab 2 report, The Isolation of Red Blood Cell Membrane Proteins... 1.5 %
Lab exam.............................................................................................12%
Problem labs...................................................................................... 5%
Lab technique mark.......................................................... up to 2% of final lab
mark may be subtracted if lab conduct and performance is shown to be poor.

2. Students must pass the lab to complete the course (50%).

3. The lab exam will be held in the lab during normal lab time, starting at 2:30 pm (refer to
schedule for date). The lab exam is 1.5 h.
4. Lab reports are to be handed in as stated in schedule by 4:30 pm of that day. Hand in lab
reports through slotted filing cabinet drawer in room 414 ONLY. Instructor and
demonstrators do not accept lab reports. If handing in a lab late, 1 mark will be subtracted
for each class day late. Marked lab reports will be returned to students the next week
(exception lab 1, as section 2 students have not handed in their reports yet). A late
report will not be accepted after that report has been returned to the class.
5. Lab report marks are final unless an obvious error in addition of marks has been made.
However, if a student feels they have a legitimate complaint, please direct attention to lab
instructor.
6. Approximately two weeks prior to the lab exam, a brief outline of lab exam format and
information content will be available on website. You are responsible for fundamental
information presented during the tutorial lab. There is an example lab exam in the lab
manual appendix.
7. You must notify the lab instructor no later than two school days after the missed lab exam
of your intent to write a deferred lab exam. The deferred lab exam must be rescheduled
before the end of this term’s classes. Failure to comply will result in a zero on your lab
exam.
8. Plagiarism (copying another student’s lab report (present or previous year) or
copying published literature without citing is a violation of University regulations.
Refer to the STUDENT DISCIPLINE BY-LAW in your student handbook (rule
book) for action taken for plagiarism.
 5

LAB REPORT PRESENTATION

1. A reference file is available in the science library (1 hour reserve).

2. Lab reports must be written in pen (no pencil) or typed.

3. Number pages.

4. On the front page of the report state:

• Course name and number
• Experiment number and Title
• Group # and section #
• Individual or Group name(s). If handing in an individual report, also include lab
partners name.
• GROUP report or INDIVIDUAL report
• Date
3. Lab report information is to be presented exactly as requested in lab manual. Number
sections the same as the lab manual.

4. Lab report may be done as an individual effort or a group effort by the two students that
carried out the experiment. One report or two reports may be handed in per group. The
decision on the number of reports per group is totally dependent on members of the
group. This decision may be changed any time during the term. Therefore for each lab
report the group has the option to hand in one or two reports exclusive of what has been
done before or after that particular report. Indicate on the cover page of the report if
the report is a group report or an individual report. If handing in an individual report
also include lab partner’s name.

5. Always include a sample of each type of calculation in your lab report.

6. If a group’s data is not workable, borrow data from another group and reference. Non
workable refers to data that cannot be plotted, used for calculations or required analysis.
It does not necessarily mean the expected data.

7. Cite reference in text of lab report and record full reference at end of lab report. When
should you cite and reference. The following is a good definition of plagiarism that
explains when you should cite a reference. “The unacknowledged use of another
person’s work, in the form of original ideas, strategies, and research, as well as
another person’s writing, in the form of sentences, phases and innovative
terminology.” (Spatt1, 1983, p.438) This is done by using bracketed reference number
that you used when listing references at end of lab report or by bracketing first authors
name and date. Quote text unless you paraphrase completely in your own words. But
remember, quotes should only be a small part of your work. If you are using the name
year system, list the references alphabetically. Some examples are as follows
(McMillan2 1997):

Binder V. Hendriksen C, Kreiner S. 1985. Prognosis in Crohn’s disease - - based on

1
Spatt, B. (1983). Writing from Sources. New York: St. Martin’s Press.
2
McMillan V.E. 1997. Writing Papers in the Biological Sciences. 2nd ed. Boston:
Bedford Books: 1997. 197 p. and McMillan, V.E. 2001. Writing Papers in the Biological
Sciences. 3rd ed. Boston: Bedford Books. 123 p.
 6

results from regional patient group from county of Copenhagen. Gut 26:146-50.
Danforth DN, editor. 1982. Obstetrics and gynecology. 4th ed. Philadelphia: Harper
and Row. 1316 p.
Petter JJ. 1965. The lemurs of Madagascar. In: DeVore I, editor. Primate behavior:
field studies of monkeys and apes. New York: Holt, Rinehart and Winston. p
2920319.

If available only on the web:

Kingsolver JC, Srygley RB. Experimental analyses of body size, flight and survival in
pierid butterflies. Evol. Ecol. Res. [serial online] 2000;2:593-612. Available from:
Colgate University online catalog. Accessed 2000 Oct 3.

8. Personal or Professional Electronic sources:

Cite in-text by putting the following in parentheses, author’s last name or file name (if
no author’s name is available) and publication date or the date of access (if no
publication date is available).
At the end of report list
(i) author or organization
(ii) publication date or date last revised
(iii) title of Web site
(iv) URL site in angle brackets
(v) the date accessed.

Cameron, L. 60.344 Microbial Physiology Lab Information

<http://www.umanitoba.ca/faculties/science/microbiology/staff/cameron/60_344.htm>.
Accessed 2002 April 12.
Table presentation
• Table number and title (legend) presented above the table body.
• Number tables using arabic numbers, even if only one table in a report.
• Include enough information in title to completely describe table, eliminating the
necessity to search elsewhere in the lab report to understand information presented in
table. Table title starts with an incomplete sentence. Additional complete sentences
may be included to adequately describe the table (this also applies to figures).
• If abbreviations are used in table, indicate what abbreviations mean as a footnote. Other
footnotes may be required to clarify material in the table.
• Like information should be in columns making it easier to view the table.
• Data in columns should be listed under the centre of each heading. Align decimal
points and dashes. If a number value is less than 1 always include zero before the
decimal.
• Column or Row headings should be complete and self explanatory. A heading is a
separate entity from the title. It cannot be assumed information given in the title is
adequate for a heading. The unit of measurement should only be included in the
heading, not in column data.
• Group related column headings under larger headings.
• If information is the same for each column or row do not include but treat as a footnote.
• Make the table as concise as possible but include all necessary information. For
example, any constant experimental conditions that would change the data presented.
• Tables should be properly set up with a straight edge. Horizontal lines must be
included but it not necessary to always include vertical lines.
Figure presentation (graphs, diagrams, photographs, films)
• Figures are to be numbered separate from tables, using arabic numbers. Include figure
number even if only one figure.
• Figure number and figure legend should be presented below the graph. The figure
 7

legend, like the table, starts with an incomplete sentence describing the graph. For
example, do not repeat just the labels of the x- and y-axis but present in a descriptive
manner. Additional sentences should be included if additional information is required to
completely describe figure, for example, any constant experimental conditions that affect
the data presented.
• All diagrams, photographs, and films are figures and should be completely labelled.
• For figures of graphs, there is one dependent variable plotted and one or more
independent variables plotted. The dependent variable is a function of the independent
variable. It is accepted practise to plot the independent variable on the x-axis and the
dependent variable on the y-axis. For example the measurement of absorbance
(dependent) with increasing concentration of protein (independent). The size of the graph
should fit the plot(s). The axis should not necessarily start at zero. Place graph
completely within graph grid, this includes axis labels and legend. The overall size of
graph should not be too large but should not be so small that information is obscured.
The graph must be completely labelled (always include units). Use different symbols for
each plot (not different coloured pens) on a graph. If more than one plot, explain symbols
in legend or in a key included in the body of the graph. Graph plots can be drawn in a
number of ways (this depends on the plot): (a) best fit straight line, (b) join each point
with a straight line, and (c) use a flexible curve ruler or french curve. Do not drawn a free
hand line.

Note: When writing your lab reports you are frequently requested to present both a table and a
figure for a given set of data, similar to keeping a research journal. This is not the accepted
practice for papers published in journals or books. Usually either a table or a figure is presented
for a given set of data and depending on nature of data, it may only be summarized in the text.
How do you make a choice of data presentation? The aim is to effectively and efficiently
demonstrate what you want to show, for example, correlations, comparisons, pattern, trends, etc
(McMillan 1997).

REFERENCE

McMillan V.E. 1997. Writing Papers in the Biological Sciences. 2nd ed. Boston: Bedford
Books: 1997. 197 p. and McMillan, V.E. 2001. Writing Papers in the Biological Sciences. 3rd
ed. Boston: Bedford Books. 123 p.
 8

LAB STANDARD OPERATIONS PROCEDURE (SOP)

Bacteria are not used in the Membrane and Cellular Biochemistry Lab. Other lab courses do use
bacteria in this lab. Animal blood and tissue products are used in your lab. Handle with care.
Wear gloves.

Bench area: Wash bench area before and after use with AIRx109.

Personal safety: You must wear a lab coat. Wear coat only in the lab, transport separately outside of
the lab (in a plastic bag). Wash hands with antibacterial soap before leaving the lab. No eating or
drinking in the lab. Use aseptic technique for transfer of bacteria. This is to protect yourself as
much as to ensure the purity of your culture. Protect hands with gloves and eyes with glasses when
needed. The gloves provided in the lab are to be disposed of after use.

Biohazards: Know biosafety risk groups. Handle all cultures as potential pathogens. Never mouth
pipette. Always use a pro-pipette. If you spill a culture, cover the spill with paper towels. Pour
AIRx109 over the towels to saturate. Gather up soaked towels and discard. Wipe area to dryness
with fresh paper towels. Wash hands with soap and water. Place cultures on discard trolley. All
cultures are autoclaved before disposing. Dispose of eppendorf tubesa in petri plate containers.
Dispose of pipetman tipsa in clear plastic lined basins along with glass or plastic Pasteur pipets,
broken glassware, glass slides, brittle plastic objects, metal objectsa (not needles or blades). Bacteria
dilutions may to be poured down the sink and the tubes rinsed before placing on the discard trolley.
Rinse sink with lots of water.
a
due to the multi-use nature of the teaching lab, all eppendorf tubes, pipetman tips, Pasteur pipets,
brittle plastic or metal objects will be treated the same as similar items contaminated with
microorganisms.
When handling level 2 microorganisms you must wear disposable gloves, make sure any cuts on
your hands are covered with a bandage, and be aware of the possibility of bacteria aerosol when you
flame your loop.

Glassware (unbroken): Remove tape and pen markings (use alcohol) from glassware before
placing on discard trolley. Used glassware should be rinsed and placed on the discard trolley.
Rinsed test tubes should be placed in tray provided on the discard trolley. Used glass pipettes should
be placed in pipette holders.

Petri plate culture and non-sharps solid culture material disposal: use covered plastic containers
lined with clear plastic bags for contaminated petri dishes or any bacteria contaminated solid non-
sharps material (eppendorf tubes, API strips, antibiotic strips, microtitration plates, etc)

Hazardous material disposal: Examples: radioactive material, ethidium bromide, sovents, etc. The
lab demonstrator will instruct proper disposal methods for labs that contain hazardous materials.
These materials must be disposed of in appropriately labelled containers and disposed via the safety
office. Use fumehood when recommended. A MSDS binder available in lab gives information on
all hazardous materials used in the lab. Use extreme care with flammable solvents. Alcohol used to
flame spread rod should never be positioned within 40 cm of flame. Never put a very hot spread rod
into a beaker of alcohol. The alcohol may catch fire. Many of the immunochemicals are preserved
in 0.1% Na azide...handle with gloved hands. Handle caustic (acids and bases) solutions with care.
Never discard an acid or base greater than one molar down the sink. Discard in labelled glass
containers provided. Use lots of water when discard caustic solutions (< 1M). These materials are
disposed of through the university safety office. Never pour solvents down the sink (eg. phenol,
ether, chloroform, etc). Discard in labelled containers provided.

Sharps disposal: Dispose of all sharps (needles, syringes, razors, scalpel blades) in specified
container. Dispose of syringe with needle attached - do not take apart. Do not replace the needle cap
before disposing (high frequency of accidents occur when replacing cap). Sharp’s containers are
autoclaved before disposing. .
 9

Broken glass disposal: Dispose of broken glass in labelled plastic containers lined with clear
plastic. Transferred to boxes before discarding.

Know location: Exits, fire extinguisher, eye wash, sink shower, and first aid kit. This
information is given in the first pre-lab.

Equipment operation: Know how to operate equipment before use. DO NOT use equipment
unless you know exactly how to operate the equipment. The demonstrator is always available to
assist. Please follow instructions in appendix for proper clean up of Spectronic 20D. Ensure the
spec tubes are thoroughly washed and rinsed with distilled water before replacing in rack upside
down as you (hopefully) found the tubes.

Leave your bench area clean All equipment and supplies should be returned to original
location.

LABORATORY BIOSAFETY GUIDE

No bacteria are used in this lab. It is still important that you follow standard operation
procedures, SOP (see above).

The University of Manitoba Biosafety Guide (Feb 2000) and Health Canada Laboratory
Biosafety Guidelines booklets are available in your lab. Biosafety information is also available at
the Health Canada websites:
Guidelines: http://www.hc-sc.gc.ca/pphb-dgspsp/ols-bsl/lbg-ldmbl/index.html Health
Canada http://www.umanitoba.ca/campus/health_and_safety/
MSDS (infectious agents):http://www.hc-sc.gc.ca/pphb-dgspsp/msds-ftss/index.html

There is no listing of level 1 agents in the guidelines or MSDS pamphlets

Risk group 1 bacteria are low individual and community risk and are unlikely to cause disease
in healthy workers.
Risk group 2 bacteria are moderate individual risk and limited community risk. Bacteria in this
group can cause human or animal disease but are unlikely to infect healthy laboratory workers.
Effective treatment is available. Risk of spreading is limited.

CONTAINMENT LEVEL 1 (UM biosafety guide p. 11)

• microbiology lab with washable walls, countertops and hand wash sink
• established safe laboratory practices (hand washing and disinfection of countertops)
• general WHMIS safety training
• UM lab registration

CONTAINMENT LEVEL 2 (UM biosafety guide p.11)

• all of level 1 specifications
• biosafety permit
• biological safety cabinet (not required)
• biohazard sigage
• a written standard operations procedure
• MSDS for the infectious agent
 10

WHMIS

The Workplace Hazardous Materials Information System (WHMIS) is a system for safe
management of hazardous materials. WHMIS is legislated by both the federal and provincial
governments.

Under WHMIS legislation, laboratories are considered to be a workplace, and students are
workers. By law, all workers must be familiar with the basic elements of the WHMIS system.

The WHMIS program includes:

1. Cautionary labels on containers of controlled products. Consumer products, explosives,
cosmetics, drugs and foods, radioactive materials, and pest control products are regulated
separately, under different legislation.
2. Provision of a Material Safety Data Sheet (MSDS) for each controlled product.
3. A worker education program.

1. A. SUPPLIER LABELS

Controlled products must have a label of prescribed design which includes the following
information:
PRODUCT IDENTIFIER - trade name or chemical name
SUPPLIER IDENTIFIER - supplier's name and address
MSDS REFERENCE - usually, "See MSDS supplied"
HAZARD SYMBOL - (see illustration on next page)
RISK PHRASES - describes nature of hazards
PRECAUTIONARY MEASURES
FIRST AID MEASURES

B. WORKPLACE LABELS

All material dispensed in a workplace container must be labelled with the Product Name,
Precautionary Measures (simplified) and Reference to Availability of MSDS.

2. MSDS

Individual course MSDS are located in a binder in your lab (Room 201 binder located in 204).
The main MSDS binders are located in the Microbiology preparation room, 307/309 Buller.
MSDS are also available on the local area computer network (see your demonstrator, if
necessary).

The MSDS will provide: relevant technical information on the substance, chemical hazard data,
control measures, accident prevention information, handling, storage and disposal procedures,
and emergency procedures to follow in the event of an accident.

3. SAFETY

The Laboratory Supervisor will provide information on the location and use of safety equipment,
and emergency procedures.
 11

Table of WHMIS LABELLING

 12

EXPERIMENT 1

THE ISOLATION AND QUANTITATION OF UBIQUINONE FROM BOVINE HEART

The quinone, ubiquinone (Coenzyme Q10) is a membrane associated component of the

electron transport system. Ubiquinone is the only non-protein component of the electron
transport system. This allows ubiquinone to move freely through the lipid membrane.
Ubiquinone accepts hydrogen atoms and donates electrons. This feature is used experimentally to
determine the amount of ubiquinone present in a sample.

O OH

CH3O CH3 CH3O CH3

CH3O (CH2CH=CCH2)10H CH3O (CH2CH=CCH2)10H

O OH
CH3 CH3

Oxidized Form of Ubiquinone Reduced Form of Ubiquinone

Figure 1. Structure of oxidized (ubiquinone -10) and reduced Coenzyme Q (ubiquinol -10). Coenzyme Q is
composed of a quinone component with a isoprenoid (5 C unit) side chain. Ubiquinone has 10 isoprenoid units in
the side chain. Complete oxidation requires two hydrogen atoms.

In this experiment there are four steps involved in the isolation and quantitation of ubiquinone;
saponification, extraction/evaporation, thin layer chromatography, and wavelength scanning
spectrophotometric analysis.

Thin Layer Chromatography

Thin layer chromatography involves three components: a stationary phase, a mobile

phase (solvent), and a sample dissolved in a liquid. The basic principle of thin layer
chromatography is the same as column chromatography and paper chromatography. The
stationary phase is spread as a thin layer on a glass plate or plastic sheet. The mobile phase
(solvent) travels upwards through the thin layer by capillary action. As column chromatography,
the process is based on partition equilibrium between the mobile phase and the stationary phase
(more polar) as these phases compete for the components of the compound. Once the compound
is spotted on the plate the solvent moves over the adsorbed spot shifting the equilibrium and the
compound desorbs. This equilibrium depends on the compound solubility in the solvent. This
re-dissolved material is then carried to the edge of the spot where fresh adsorbent is contacted.
This process repeats itself during the development of the TLC plate until each component of the
compound has migrated a characteristic distance depending on its solubility in the solvent with
the most soluble components moving the fastest. Each component has a characteristic Rf value,
although Rf values are dependent on carefully controlled experimental conditions.
 13

distance travelled by spot from origin

Rf = distance travelled by the solvent from the origin

It is important to keep application of sample to the TLC plate as small a spot as possible
to obtain good separation of compound components. If irregular spot shapes (tailing) occur, it
may be due to overloading.

Wavelength Scanning Spectrophotometer

The Ultraspec 4000 spectrophotometer

Light from either visible (tungsten lamp) or UV (deuterium lamp - less than 340 nm) enters the
monochromator via an inlet slit and then passes through one of several filters. The filter
selection depends on the wavelength. The filtered light is directed onto the holographic grating
which produces light of a selected wavelength. The selected wavelength of light then exits the
monochromator via an exit slit. By the use of mirrors the single wavelength light passes through
the sample and a de-focusing lens to a solid state detector unit. The resulting signal is amplified
and displayed via a computer link.

SWIFT software
The spectrophotometer is controlled by Windows 95 SWIFT software package installed on an
attached computer. This software package manipulates and analyzes the data, then the data can
be printed.

PROCEDURE

Precautions

1. Ether is extremely flammable. No burners. Even a spark generated from friction

may ignite ether. Work as much as possible in the fumehood when handling ether.
2. Wear disposable gloves, especially for saponification step. Alkali will burn through the
gloves but it gives you some time.
3. Caution must be exercised when refluxing beef as procedure is carried out in the presence
of strong alkali.
4. Chromatography is performed in the fume hood as solvent is 20% diisopropyl ether in
chloroform.
5. If you are allergic to ether, please inform the demonstrator.

Note: It is IMPORTANT to record all volumes and dilutions where appropriate as this is a
quantitative measurement of ubiquinone.

WEEK 1

Come to lab prepared to answer the following questions.

1. What is an aqueous solution?
2. What is an organic solution?
3. What type of solution is ether? organic or aqueous?
4. What type of solution is your beef heart mixture (beef heart, KOH, methanol)?
5. After shaking ether with saponified beef heart mixture in the separatory funnel, two layers
are formed. What is the top layer? What is the bottom layer. Explain why. Hint: think
relative densities.
 14

Saponification

1. Each group will receive a pre-weighed quantity of bovine heart tissue (connective tissue
and fat removed). Record weight. Using scissors, cut heart tissue into small pieces
(approx 0.5 cm by 0.5 cm) and place in a 250 or 300 ml glass beaker. The sample will be
homogenized by the demonstrator. For homogenizing you will require the cut beef heart
tissue and 100 ml -20oC methanol. Add approximately 1 g pyrogallol (lots) to your

REFLUX APPARATUS

rubber tubing to sink (OUT)

condenser

clamp

stand

rubber tubing from tap (IN)

clamp

waterbath (80 - 90
oC)

Figure 2: Reflux apparatus diagram for saponification of beef heart.

methanol just before homogenizing. Shake vigorously to dissolve. Then pour 50 ml into beaker
containing cut bovine tissue. Homogenize (Polytron) for 15 to 30 sec or until a homogenous
mixture is obtained.
 15

2. After homogenizing, add remaining methanol and 25 ml 60% KOH, mix and pour entire
mixture into the 1 liter round bottom flask. Clamp to stand such that the mixture is
submerged in the waterbath. Attach condenser (also clamped to stand). Complete reflux
set up as shown in Figure 2. Turn on water to condenser slowly and maintain at low
water pressure (just enough to keep the condenser full of water).

3. Reflux in a simmering (80-90oC) water bath for 45 min. Saponification should be

monitored to prevent too rapid boiling as the mixture may bubble out the top of the reflux
apparatus.

4. Rapidly cool to 25oC by adding 75 ml ice cold distilled water. Place on ice if necessary.
Proceed to the next step when mixture is at room temperature.

Extraction and Evaporation

5. When the mixture is at room temperature, transfer it into a separatory funnel and add 67
ml absolute ether plus 33 ml petroleum ether (add ether in fumehood). With the stopper
in place, carefully mix the organic and aqueous phases by turning up and down. Do not
shake the separatory funnel as this will create a froth causing a middle phase which is an
emulsion of the upper and lower layer. This emulsion layer is almost impossible to
remove.
It is EXTREMELY important that you release the pressure from the funnel. Periodically
invert the funnel with the stopper in place and open the release valve. When releasing
pressure, make sure the spout is not pointing at anyone. Release of pressure must be done
in the fumehood.

6. Collect the organic phase and repeat the extraction of the aqueous phase a second time
with 67 ml absolute ether and 33 ml petroleum ether. Pool the two ether extracts and
measure the volume. When considering which phase is the organic phase, upper or
lower, remember to consider the density of ether compared to water. If not sure which
layer to collect be sure to ask the teaching assistant.

7. Transfer to cleaned separatory funnel and wash the pooled extract twice with saline
(0.85% NaCl). The volume of saline added depends on volume available in separatory
funnel that still allows mixing in separatory funnel. Again with the stopper in place,
carefully mix the organic and aqueous phases by turning up and down to prevent the
formation of an emulsion layer. If a middle emulsion layer does occur, add a spatula of
NaCl and invert to mix - this may help. It may take 15 minutes for the phases to separate
(especially last saline wash). If it is taking this long ask the teaching assistant for advice.
When collecting organic phase, DO NOT CONTAMINATE with any water as this will
make complete evaporation by aspiration impossible.

8. Evaporate the washed extract to dryness using the rotatory vacuum as described below
diagram.
16
 17

9. Once the sample has been evaporated to dryness resuspend in 5 ml petroleum ether and
transfer to a 4" screw capped test tube. Label tube with group number and TEST sample
only (use masking tape). The demonstrator will evaporate you sample under a stream of
nitrogen before next the lab period. Your samples will be resuspended in specified
volume, recorded on the eppendorf tube, of absolute ethanol. Record this volume. Store
at -20oC until week 2.

WEEK 2
Thin Layer Chromatography

1. The TLC chambers have been set up prior to the start of the lab and are located in
fumehood. The glass chamber contains 50-100 ml solvent (20% isopropyl ether in
chloroform). A piece of 3 MM filter paper is placed in the chamber along one side such
that the lower edge is immersed in the solvent. The sintered glass edges of lid and top of
the chamber are coated with Vaseline allowing the lid to fit tightly. The chamber is
equilibrated allowing the atmosphere to saturate with solvent (at least 1 h).

2. The TLC plates (silica gel) have been activated by drying in an oven for 30 min at 100oC.
3. Prepare thin layer plates by lightly marking two spots 3 cm from one end and each 2 cm
from a side. Do not press so hard on the pencil that you remove the silica gel. Spot 5 :l
of your TEST sample using a micropipet. Note: If sample has precipitate, the sample
should dissolve upon warming - hold vial under running hot water. Caution: Do not heat
too much or sample will evaporate in the pipette tip before you have a chance to spot on
gel. It is important to keep the size of the spot small (2-4 mm). Allow the spot to dry
and repeat process until a total of 50 :l sample applied. Record amount spotted.
Return remaining TEST sample to demonstrator.

4. Each group will be given an unknown sample of commercially pure ubiquinone (notated
as STANDARD sample). Spot 50 :l on TLC plate in 5 :l aliquots as done in step 3.
Return remaining STANDARD sample to demonstrator.

5. Develop the TLC plate in a solvent consisting of 20% isopropyl ether in chloroform. The
process of placing the TLC plate into the chamber should be done quickly but with care
(maintain saturated atmosphere). Remove the lid and immerse the plate into the solvent
evenly and slowly to allow the solvent and plate to contact evenly. Close chamber lid.

6. Development of the TLC plate: Remove the plates after the solvent has travelled up 3/4
of plate. In the fumehood immediately draw a line with a pencil to mark solvent front.
Measure and record distance from spot line to solvent front. Allow plates to dry in
fumehood. In your report make an illustration of the final appearance of the plate.
Record distance standard ubiquinone moved (centre of sport).

7. Scrape off that portion of the plate that corresponds to your STANDARD ubiquinone
sample (yellow in color) and bovine heart TEST sample. If the bovine heart TEST
sample does not appear yellow, scrape off area that corresponds to distance that
STANDARD sample (pure ubiquinone) has migrated. To remove the silica gel sample
from the plate use the flat end of a weighing spatula to scrape the yellow spot onto a piece
 18

of weighing paper.

8. Set up a Pasteur pipette column for each sample as shown in Figure 4. Carefully pour
silica gel containing sample into a pasteur pipette column containing glass wool. Elute
the ubiquinone from the silica column by slowly adding absolute ethanol drop by drop
(use a total volume of 3 ml absolute ethanol). The silica gel should become colorless.
Collect the eluted sample in a 4" screw capped test tube.

9. Label samples with group number and either STANDARD (pure ubiquinne) or TEST
(your beef sample) ONLY using a masking tape label. The samples will be stored at -
20oC until next week.
Note: precipitate in sample does not affect the quality of sample.

Wash with ethanol (drop by drop)

Pasteur
pipette

Silica gel containing sample

(scraped from TLC plate)
Glass wool

Collection vial

Figure 4: Pasteur pipette column for concentrated beef heart ether extract.
 19

WEEK 3

Wavelength Scanning Spectrophotometry

[Ultrospec 4000 Spectrophotometer]

For this section, each group will be assisted by the demonstrator at assigned times.

1. Using the scanning spectrophotometer determine the absorption spectrum of the

ubiquinone sample (1 ml) between 225 and 350 nm. This is the spectrum of oxidized
ubiquinone.
Optional procedure: Just before measuring oxidized form of ubiquinone add 2 :l 5%
KOH in 95% ethanol (completely oxidizes your sample).
Note: If the OD at 275 nm is greater than 2 OD the sample should be diluted prior to
performing the scan. The TA will tell you if this is necessary.

2. Next, determine the absorption spectrum of reduced ubiquinone by adding one or two
crystals of sodium borohydride (NaBH4) to the cuvette before scanning. After the
addition of sodium borohydride, mix and wait one minute for the reaction to cease and
NaBH4 to settle out.

LAB REPORT

Data Presentation and Analysis

Include only information requested and give a sample of each different calculations.

1. a) Present a completely labelled figure of developed TLC plate.

b) Calculate the Rf value of STANDARD ubiquinone sample. Why is it difficult to
compare your Rf value to literature values. What information is obtained about
ubiquinone from Rf value? Explain the bases for your answer.

distance travelled by spot from origin

Rf = distance travelled by the solvent from the origin (running front)

2. Present completely labelled figures of the scanning spectrums for beef heart (test) and
standard ubiquinone.

3. a) Tabulate scanning spectra data for both samples (TEST and STANDARD) as shown
below.
Wavelength (nm) Absorbance (A) Difference between Spectra
()A) (Oxidized minus Reduced)
Oxidized Reduced
225
230
235
*

* Record wavelength every 5 nm for table and difference spectra.

continued . . .
 20

. b) Present figure(s) of the difference spectrum for TEST and STANDARD samples.
Note: The differences between these reading will be positive at some wavelengths and
negative at other wavelengths. If you have negative values, plot the results so that you
can have both minus and plus values on the graph.

4. a) Present a table of test and standard ubiquinone sample volumes/weights that are
required for calculation of ubiquinone.

b) Determine the amount of ubiquinone (moles/g) in beef heart muscle (TEST) and
determine the original concentration of STANDARD (mM).

This determination is based on the difference in absorbance at 275 nm between the

oxidized and reduced forms of ubiquinone. Using purified compound it has been
determined that a 1 mM solution of ubiquinone will show a change in absorbance of 12.5
absorbance units between the oxidized and reduced forms at 275 nm in a 1 cm pathway
cuvette. Therefore, you can calculate the mM concentration of ubiquinone in the cuvette
by subtracting the absorbance of the reduced sample at 275 nm from that of the oxidized
sample at 275 nm and dividing the resulting number by 12.5.

For the TEST sample determine the total number of moles of ubiquinone in your sample.
Express this in moles/g wet weight of beef heart.

For the STANDARD sample, determine the number of moles spotted on the TLC. Then
determine the original concentration of STANDARD pure ubiquinone sample (mM)
handed out in class.

Questions:
Note: Wherever applicable, remember to include reference used to answer questions.

1. Explain why the saponification step is included. What precautionary measures (two)
were taken during saponification? Explain why.

2. After evaporation and resuspension of the beef heart extracted sample in ethanol (prior to
applying to the TLC), are there other lipids present? Explain why/why not.

3. Briefly outline a procedure to separate (purify) coenzyme Q10 (ubiquinone) from

contaminating coenzymes, Q6 to Q9.

4. Often the absorbance peak of an ubiquinone sample does not agree with expected. For
example, (i) a broad peak that does not reduce or (ii) the peak maximum is different. For
each case explain why this may happen. Assume experiment performed exactly as was
done in your lab except ubiquinone was extracted from bacteria.

5. Why was beef heart selected as the source of ubiquinone?

6. Outline in detail how to prepare each of the following:

(i) 500 ml 20% isopropyl ether in chloroform (v/v)
(ii) 200 ml 60% KOH (w/v)

w = weight; v = volume

7. Ask a question about the lab procedure. For example, what is the function of pyrogallol?
Questions will be collected and discussed in question lab (refer to schedule).
 21

REFERENCES

Any basic biochemistry book.

References located in your lab reference file in the Science Library.

# Reference (1 hour reserve in Science and Technology Library)

The isolation and quantitation of ubiquinone

1 Madigan, M.T., Martinko, J.M. & J. Parker. 2000. Brock Biology of Microorganisms. 9th ed. Upper Saddle
River: Prentice Hall. p 121-125.

2 Findlay, J.B.C. & W.H. Evans. 1987. Biological Membranes. A practical approach. Washington: IRL Press.
p 108-113.

3 Redfearn ER. 1967. Isolation and Determination of Ubiquinone. In: Methods in Enzymology. XI:381-384.

4 Brodie AF. 1963. Isolation and Photoinactivation of Quinone Coenzymes. In: Methods in Enzymology.
VI:295-309.

5 Sawyer, Heineman, Beebe 1984. High Performance Liquid Chromatography (HPLC). Chemistry
Experiments for Instrument Labs. p .344-347

6 Sawyer, Heineman, Beebe 1984. Thin-Layer Chromatography (TLC) Chemistry Experiments for Instrument
Labs. p 387-394.

6a Crane, F.L., Barr, R. 1976. Determination of Ubiquinone. In: Meth. Enz. XVIII Part C:137-181.
 22

EXPERIMENT 2 RED BLOOD CELL MEMBRANE PROTEINS

Red Blood Cell Membrane Proteins

In the early days of SDS-PAGE virtually nothing was known of the protein components of the
membrane and consequently they were identified by numerical systems. The system was established by
Fairbanks et al in 19713 where eight major polypeptide bands were found, numbered from 1 to 8:
highest to the lowest molecular weight.
This was an oversimplification of the polypeptide pattern. As techniques were refined other
polypeptide bands appeared, refer to figure4 below. The system was expanded by introduction of a
binomial system e.g. bands between 2 and 3 were designated 2.1, 2.2, 2.3 etc. Briefly the following
bands have been identified: bands 1 and 2 are spectrin polypeptides, 2.1 is syndein, 2.2, and 2.3
proteolytic breakdown products of 2.1, band 3 is the anion channel protein, band 5 is actin and 6 is
glyceraldehyde-3-phosphate dehydrogenase. The major glycoproteins are glycophorins PAS 1, PAS 2,
PAS 3 and PAS 4. It is probable that PAS 1 and PAS 2 represent the dimeric ("2) and monomeric (")
forms of the same major red blood cell glycoprotein, glycophorin A.

3
Fairbanks, G, Steck, TL, Wallace, DFH. 1971. Biochemistry 10, 2606-2617.
4
Thompson, S, Maddy, AH. 1982. Gel electrophoresis of erythocyte membrane proteins. In: Ellory, CJC,
Young, JD, eds, Red Cell Membranes - A methodological approach. New York: Academic Press, Inc. p 67-75, 92-
93.
 23

Protein Determination
Both the modified Lowry and Bradford procedures are used to determine membrane protein
concentration.

The modified Lowry procedure5 is used to determine total protein concentration of membrane
samples. Membrane protein samples are assayed directly without prior membrane solubilization
by adding sodium dodecyl sulfate (SDS) (modification) to the Lowry alkaline reagent. Another
modification is that copper tartrate concentration is increased to allow quantitation of protein in
the presence of interfering buffer components.
Under alkaline conditions, copper (Cu2+) binds peptide bonds. Upon binding the copper is
reduced to Cu+. Folin-Ciocalteu reagent (Stoscheck, 1990) reacts with the bound reduced copper
and the R groups of tyrosine, typtophan, and cysteine. The Folin-Ciocalteu reagent is slowly
reduced to molybdenum/tungsten blue. The color produced is directly proportional to the amount
of membrane protein present.

The Bradford procedure6 a protein determination method, is based on the binding of the dye,
Coomaissie Brillant Blue G-250 (Xylene Cyanine Brilliant G), by protein. The dye mainly
interacts with arginine residues but also interacts somewhat with histidine, lysine, tyrosine,
tryptophan and phenylalanine residues. Coomaissie Brillant Blue G-250 exists in two forms, red
and blue. Protein bound Coomaisse blue is blue (maximum absorption at 595 nm) and unbound
Coomaissie blue is red (maximum absorption at 465 nm). Color production is directly
proportional to protein concentration.

SDS Polyacrylamide Gel Electrophoresis

A convenient method for quantitating and characterizing a protein is electrophoresis in an

acrylamide gel. Electrophoresis procedures are rapid and detection of the proteins in the gel by staining
or autoradiography is convenient and sensitive.
Electrophoretic migration rates have a fairly predictable relation to the molecular weight of
proteins if the proteins are dissociated and denatured with sodium dodecyl sulfate (SDS) before and
during electrophoresis7 Most proteins bind a fixed amount of SDS per amino acid; as a result, proteins
acquire a fixed charge-to-mass ratio and differ only in their size. Electrophoresis of such proteins are
then separates (almost) according to size.
A convenient and sensitive protein stain for all-around use in polyacrylamide gel electrophoresis
is Coomassie Brilliant Blue R250. Coomassie Brilliant Blue R250 forms electrostatic bonds with the
amino groups and non-covalent bonds with non-polar regions (hydrophobic interactions) of the protein8.
This dye exhibits good linear dependence of color intensity on protein concentration within a wide
range. As little as 0.1 :g protein per band can be detected8.
When glycoproteins are stained with Coomassie Blue, the intensity of the staining is not
proportional to amount of protein present. Consequently it may appear that a glycoprotein is present in
relatively small amounts when it is not, but when stained with a specific glycoprotein stain the intensity
of stain is proportional to the amount of protein present in the band. The modified periodic acid-Schiff
(PAS) technique gives good results when staining glycoproteins.

5
Markwell, MAK, Haas, SM, Bieber, LL, Tolbert, NE. 1978. Anal. Biochem. 87:206-.
6
Bradford, MM. 1976. A rapid and sensitive method for the quantitation of microgram quantaities of
protein utilizing the principle of protein-dye binding.
7
Weber, K, Osborn, M. 1969 J. Biol. Chem. 244: 5074-.
8
Thompson, S, Maddy, AH. 1982. Gel Electrophoresis of Erythrocyte Membrane Proteins. In: Ellory CJC,
Young JD, eds., Red cell membranes - A methodological approach. New York: Academic Press, Inc. p 29,30
 24

PROCEDURE

Note: Certain sections of the lab will be performed by the demonstrator. Students are responsible for all
sections for the lab exam.

WEEK 1

Part I: Preparation of red blood cell membranes

1. Keep samples on ice throughout experiment (when not handling).

2. Each group will start with approximately 3 ml packed sheep’s red blood cells in a plastic screw
capped centrifuge tube (prepared prior to start of lab). Add approximately 30 ml PBS (contains
150 mM NaCl) and resuspend. Note: The cells are easier to resuspend if first gently mixed after
a small addition of buffer, then brought up to volume. Mix by inverting and swirling but not too
vigorous.

3. Centrifuge at 4000 rpm for 10 min at 4oC. The red blood cell pellet is a “loose” pellet. Hold
your tube at the same angle it was in the centrifuge and move carefully to your bench. If your
pellet is disturbed you MUST repeat the centrifugation step so take care. Immediately, aspirate
off the supernatant with a water aspirator that has a pipet tip attached. Gradually move the
aspirator tip down the supernatant as it is aspirated off and keep the tip away from the pellet.
Remember you are still hold the tube at a slight angle. Keep your eye on the pellet during
aspiration. Likely, you will not be able to completely remove the supernatant or you will lose
your pellet down the sink.

4. Hemolysis is performed by adding approximately 30 ml 5P8 (contains no NaCl). Replace screw

cap and mix vigorously by inverting. The resulting ghosts (membranes) are pelleted by
centrifuging at 20000 rpm for 10 min at 4oC. Again, the pellet (membrane) is “loose”. Where is
your pellet located when you removed your tube from the centrifuge? Take the same precautions
with the centrifuged sample as step 3.

5. Remove the supernatant by aspiration. The pellet is quite loose, so care is needed. STOP
removing supernatant when pellet starts to move even if some supernatant remains. The
centrifuge tube is then tipped and rotated on its axis so that the "loosely" packed ghosts slide
away from a hard "button" pellet, usually pink. This pellet is rich in proteases and should be
removed using a swab stick.

6. Wash the membranes with 5P8 buffer by adding 40 ml 5P8 buffer, mixing, centrifuging at 14000
rpm for 10 min at 4oC, and removing supernatant by aspiration. As you are never able to
completely remove the supernatant the pellet should be in 100 to 200 :l 5P8. It is okay if the
sample remains slightly pink (contaminated with un-lysed cells and haemoglobin) as this does
not interfere with polyacrylamide gel electrophoresis. Measure approximate volume with a
micropipet and adjust to a minimum volume of 100 :l with 5P8 if necessary. Transfer the
membrane sample to an eppendorf tube and clearly label with groups number (use masking tape
to label).
7. Place your sample on ice.

Part III: Protein Determination by Bradford Method

1. Set up the following reaction tubes:

(1) Standard curve: Set up reaction test tubes IN DUPLICATE for different
concentrations of BSA (bovine serum albumin) standard. Using a stock solution of 1
mg/ml BSA, add 10, 20, 40, 60, 80, and 100 :g BSA. Bring volume to 100 :l with
 25

distilled water.
(2) Blank: 100 :l distilled water.
(3) Sample: Prepare duplicates containing 4 :l and 10 :l membrane protein sample.
Bring volume up to 100 :l with distilled water.

2. Add 125 :l 2 M NaOH. Mix. Why are you adding NaOH?

3. Add 5 ml Bradford solution to each tube and mix by vortexing.

4. After 5 min but before 1 hour, read absorbance at 590 nm using spectronic 20D .
Note: timed intervals are not necessary as the color is stable for 1 hour.

5. Hand in sample to demonstrator. The samples will be stored at -20oC until next week.

WEEK 2

Part III: Protein Determination by Modified Lowry Method

1. Set up the following reaction tubes:
(1) Standard curve: Set up reaction test tubes IN DUPLICATE for different
concentrations of BSA (bovine serum albumin) standard. Using a stock solution of 1
mg/ml BSA, add 10, 20, 40, 60, 80, and 100 :g BSA. Bring volume to 100 :l with
distilled water.
(2) Blank: 100 :l distilled water.
(3) Sample: Prepare duplicates containing 4 :l and 10 :l membrane protein sample.
Bring volume up to 100 :l with distilled water.

2. At timed intervals add 3 ml reagent C to each tube and incubate at room temperature for 10 min.
Reagent C is prepared by adding 0.5 ml reagent B to 50 ml reagent A and mixing just prior to
use.
Note: If Reagent A is precipitated, put under running hot water to dissolve.

3. At appropriate timed intervals, add 0.3 ml Folin-Ciocalteu reagent D (already diluted) to each
tube and vigorously mix the contents immediately after addition. Immediate vigorous mixing
must be done for reproducible results as the Folin reagent is active for a very short time after
addition.
4. Incubate the tubes for 30 min at room temperature.

5. At appropriate timed intervals, read absorbance at 590 nma using spectronic 20D. Timed
intervals are not required if all samples are read within 10 minutes of each other. It is important
that you have no bubbles as bubbles give inaccurate high readings.
a
protein is read at 590 nm due to the spec20D wavelength range limitation(maximum = 600 nm).
Generally the blue chromophore is measured at 660 nm and has a peak maximum at 750 nm.

Part IV: Polyacrylamide Gel Electrophoresis

A. Casting, Gel Asssembly, Sample Loading, Gel Running

1. The gel electrophoresis apparatus will be set up by the demonstrator during the lab period. Make
sure you understand the gel electrophoresis apparatus operation, Fig 7. Prior to start of lab the
gel slabs have been prepared by demonstrator in the following manner: a 12 % gel solution was
poured into clamped glass plate assembly to 1 cm below the bottom of comb teeth (pre-measured
and marked). Immediately a small amount of water or butanol was carefully layered on the
surface to ensure a flat surface. Allowed to solidify for a minimum of 1 hour at room
temperature. The water or butanol was removed. The stacking gel poured, well maker comb
aligned correctly and stacking gel allowed to solidify for 30 – 45 min. The comb was carefully
removed and the wells were rinsed with water. The gel electrophoresis apparatus was partially
assembled.
 26

2. Put 20 :l red blood cell membranes in an eppendorf and add 5 :l sample buffer. Just before
loading sample on gel, heat your sample at 95oC for 4 min. Cool to room temperature. Also heat
the prepared molecular weight standard (already contains loading buffer).

3. Flush wells out with electrophoresis running buffer. Add approximately 10-15 :l sample (or
maximum volume). Ideally 10 :g protein should be loaded per well.
4. Load 7 :l of prepared protein molecular weight standard; Benchmark Protein™ Ladder (figure
9).

5. Complete set up of electrophoresis unit. Apply 200 volts for 45 min run (when the bromophenol
blue tracking dye is 0.5 cm from the bottom ).
Notes:
(i) Safety for operation of Mini- Protean® II Electrophoresis Cell
Maximum voltage limit = 600 voluts
Maximum power limit = 15 watts
maximum ambient temperature limit = 50oC
(ii)Frequently there is a question on the lab exam related to the setup and running of your SDS-PAGE.
Be sure you understand the operation of the protein gel electrophoresis apparatus that is used in your
lab. If necessary, the Mini- Protean® II Electrophoresis Cell Instruction Manual is on reserve in the lab
reference folder. It is best to observe setup and operation in your lab. Ask the demonstrators questions
on anything you do not understand related to the setup and operation of Mini- Protean® II
Electrophoresis Cell.

Remaining steps performed by demonstrator.

6. After electrophoresis is complete, turn off, completely disassemble the apparatus and remove one
glass plate. Float the gel off the remaining plate by submerging the glass plate containing the gel,
gel side down, under the fixative solution. Agitate gently until the gel separates from the glass
plate. Or gel may be removed directly in the Coomassie blue staining solution.

B. Gel Staining

Coomassie Blue R250 (Protein): Put gel in the stirring container containing 300 ml Coomassie blue
staining solution. Stir at room temperature for 1 hour. Pour off dye and save (can be reused). Destain
with several changes of destaining solution (usually 1 to 3 hours). Photograph gel before or after drying.
 27

C. Gel Processing

The BioRad Gel Doc 1000 unit (consists of enclosed box with camera positioned on top of the box) is
used to photograph the SDS-PAGE gel. The gel is placed in the box over a white light
transilluminator. The camera view is analysed by an attached computer using the software package,
Analyst Program. The file is saved as a .tif file, edited using CorelDraw software package, and
printed. OR scanned as a .jpg file and printed. As soon as possible the gel picture required for lab
report will be available on the website.
 28

Leqd to power pack Leqd to power pack

-
Negative electrode
+
Postive electrode
Lid must be attached when running the gel.

Upper (centre) buffer chamber

Polyacrylamide gel sandwiched between

two glass plate, one longer than the other. The
shorter glass plate faces inwards to upper buffer
and accessible to wells made in the gel. The wells
are open only to the centre (upper) buffer chamber.
The lower buffer is only in contact with the bottom of gel. Holder structure
Current must pass through the gel carrying the negatively of gels in glass
charged proteins. plates - must have
Negative electrode in centre (upper) buffer chamber both holders
Lower in place to run
Buffer gel to form upper
Direction of Protein Movement
buffer chamber.
This structure also
directs the electrodes
to either the upper
or lower buffer chamber.

Postive electrode in outer (lower) buffer chamber

Longer glass plate. Pipette tip adding

sample but only
after holder is
clamped to
electrophoresis
Sample wells formed in gel.. apparatus
containing buffer.
Demonstration
here only, not
actual time of
loading sample.

Plastic Shorter glass plate.

spacer
Plastic
band. spacer
band.

Gel is poured between two glass plates separated by spacer bands. A comb is used to make wells in
the gel. The glass plates are clamped into a holder when the gel is poured and allowed to harden. The clamped
gel is then attached to the centre structure of the gel electrophoresis apparatus such that the shorter glassplate
faces inwards. Sample is loaded after attaching glass plates to main electrophoresis
apparatus that contains both upper (centre) and lower buffer. Pipette tip is lowered through upper buffer
to top of specific well and the sample is released. Sample sinks to the bottom of the well due to the density of the
sample.
Figure 7. Mini-PROTEAN II Electrophoresis Apparatus.
 29

LAB REPORT
Notes:
(i) Include only information requested and give a sample of each calculations type.
(ii) Wherever applicable, remember to include references used to write lab report.

Data analysis
1. Protein Determination:
a) In one table, record spectronic 20D absorbance readings for both methods; Lowry and
Bradford.

b) For each method, present a separate figure for the protein standard curve of absorbance versus
:g BSA. Your plots should go through zero. Only when interference is caused by some
component of the assay does the plot not go through zero. If this happens to your data, it is
acceptable not to draw the plot through zero. But you must consider only the linear part of your
data when drawing the best fit line. Often with increasing BSA concentration, the slope will no
longer be linear but start to flatten9. If this happens, do not consider these points when drawing
the best fit line.

c) Determine the concentration of your membrane protein (mg/ml) sample using the slope of
standard curve for each protein determination method.
Note: If you do not know how to use the slope of your standard curve to calculate the
concentration of your sample, find out. This question is frequently asked on your lab exam with
only the slope of standard curve given in the question.

d) Are your protein concentration values similar for Lowry and Bradford? Does the
modification of the Lowry method for protein determination by Markwell et al10 address the
comment by Bradford11, “the dye binding assay (Bradford) is approximately four times more
sensitive than the Lowry assay”? Explain how/how not.

2. Polyacrylamide Gel Electrophoresis (Your data):

(SDS-PAGE student data available on website as soon as processed.)
a) Present a completely labelled figure of the printout of the polyacrylamide gel containing your
group’s sample. Label the following red blood cell membrane polypeptides designated: 1, 2,
3, 4.1, 4.2, 5, 6 & 7 and Hb (hemoglobin) (Thompson & Maddy, 1982 ). Since human red blood
cell membrane proteins are very similar to sheep, figure 10-2, in Principles of Biochemistry,
1997, may help you to identify rbc membrane polypeptides 1, 2, 3, 4.1, 4.2, 5, 6 & 7.
If your group’s data is poor, select another group’s data (reference) for labelling and calculations.
OR, if available, refer to website for supplementary data.

b) Tabulate protein standard ladder data: molecular weight (KDa) and distance migrated from
well for each band.

c) Present a figure of the standard curve of molecular weight (KDa) of standards vs distance
migrated on semi-log paper (photocopy included in lab manual) or use standard graph paper
plotting log molecular weight vs distance migrated. If using standard graph paper, include log
conversion data in table form. Use all protein standards when preparing the standard curve.

9
Stoscheck, C.M. 1990. Quantitation of Protein. In: Deutscher, M.P.ed. Method in Enzymology, Vol. 182
Guide to Protein Purification. NewYork: Academic Press, Inc. p 50-68.
10
Markwell, M.K., Haas, S.M., Tolbert, N.E. and L.L. Bieber 1981. Protein determination in membrane
and Lipoportein Samples: Manual and Automated Procedures In: Lowenstein, JM, ed. Methods in Enzymology
Vol. 72 New York: Academic Press, Inc. p 296-303.
11
M.M. Bradford 1976. A rapid and Sensitive method for the quantitation of microgram quantites of
protein ultilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254.
 30

d) Using the standard curve, determine and tabulate the molecular weight of each red blood cell
membrane polypeptides designated: 1, 2, 3, 4.1, 4.2, 5, 6 & 7. Also include columns for
designation and distance migrated from well. Values may be read directly from graph paper (a
must for semi-log paper). It may not be possible to determine the molecular weight for all
polypeptides.

Questions

1. Is it possible to determine an accurate molecular weight of glycoproteins using SDS-PAGE?

Explain you answer.

2. a) What is the principle of red blood cell membrane isolation procedure used in your lab.
b) Explain how it is possible to measure membrane protein concentration by Lowry and
Brandford method in your lab, when many of the membrane proteins are embedded in the
membrane.

3. a) In your lab you used bovine serum albumin as the standard for both the Lowry and the
Bradford protein determination assay. Why was BSA selected? Would the concentration of your
sample change if gelatin was the standard? Explain why/why not.
b) Outline a procedure to determine if protein sample components interfere with protein
concentration determination.

4. Silver staining may be used in combination with Coomaissie blue staining of SDS-PAGE gel.
What is the advantage?

5. Describe what your stained gel results would look like if the following happened. Explain why.
(a) white blood cells not removed
(b) added TLCK (4-"-tosyl-L-lysylchloromethane) during isolation of red blood cell membranes
(c) voltage too high during electrophoresis of SDS gel (do not need to explain why)
(d) use 5% acrylamide instead of 12% used in the lab (do not need to explain why)

6. Is it possible to use SDS-PAGE as a purification step to isolate a biologically active membrane

protein? Explain why/why not.

7. How do you determine the theoretical protein gel exclusion molecular weight for a 12%
acrylamide gel? Use this information to comment on your molecular weight standard curve
shape.
8. Ask a question about the lab procedure. For example, what is the function of PBS (phosphate
buffered saline)? Questions will be collected and discussed in question lab (refer to schedule).
 31

3 cycle semi-logarithmic
32
 33

REFERENCES

References located in your lab reference file in the Science Library.

# Reference (1 hour reserve in Science and Technology Library)

Experiment 2: Red blood cell membrane proteins

7 Jere P. Segrest & R.L. Jackson. 1972. Molecular Weight Determination of Glycoproteins by Polyacrylamide
Gel Electrophoresis in Sodium Dodecyl Sulfate. In: Ginsburg, V. ed. Methods in Enzymology. 28: Part B. p
54-55.

8 S. Thompson & A.H. Maddy 1982. Gel Electrophoresis of Erythrocyte Membrane Proteins. In: Chapter 5 of
Text Red Cell Membranes, eds., Ellory C.J.C.& Young J.D. p 67-75.

9 Maddy, A. H. 1982. The Solubilization of Erythrocyte Membrane Proteins. In: Chapter 4 of Text Red Cell
Membranes, eds., C.J.C. Ellory & J.D. Young. p 43-63.

10 SDS polyacrylamide gel electrophoresis Instruction Manual and Related Information.

11 M.M. Bradford 1976. A Rapid and Sensitive Method for the Quantitation of Microgram Quantites of Protein
Ultilizing the Principle of Protein-Dye Binding. Analytical Biochemistry 72: 248-254.

12 Osterman, L.A. 1984. Electrophoresis of Proteins. In: Methods of Protein and Nucleic Acid Reseach.
Springer-Verlag. p30-39.

13 Markwell, M.K., Haas, S.M., Tolbert, N.E. and L.L. Bieber 1981. Protein Determination in membrane and
Lipoportein Samples: Manual and Automated Procedures In: Lowenstein, J.M. ed. Methods in Enzymology
72:296-303.

15 US/EG Bulletin 1156 REV D. 1997. Acrylamide polymerizaton - a practical approach. BIO-RAD. p 1-8.

16 Andrews AT. 1986. Electrophoresis: theory, techniques and biochemical and clinical applications. 2nd ed.
Oxford: Oxford Science Publications. p28-40

17 Stoscheck, C.M. 1990. Quantitation of Protein. In: Deutscher, M.P.ed. Method in Enzymology. NewYork:
Academic Press p 50-68.

18 Deutscher, M.P. 1990. Maintaining Protein Stability. In: Deutscher, M.P.ed. Method in Enzymology.
NewYork: Academic Press p 83-89.

19 Lehninger, A.L., Nelson, D.L. & M.M. Cox. 1993. In: Principles of Biochemistry. New York: Worth
Publishing. p 270

References not in the reference file (if necessary, for additional information).

Andrews, A.T. 1986. Electrophoresis: Theory, Techniques, and Biochemical and Clinical
Applications. 2nd edition, Oxford University Press, New York.

Steck, T.L. and Kant, J.A. 1974. Methods in Enzymology. Vol. XXXI, p. 172-3

Weber, K. and Osborn, M. 1969. J. Biol. Chem., 244: 4406 - 4412.

 34

REAGENTS

PBS: 150 mM NaCl, 5 mM sodium phosphate, pH 8.0

5P8: 5 mM sodium phosphate, pH 8.0

Lowry Reagent Composition

_________________________________________________________________________
A 2% Na2CO3, 0.4% NaOH, 0.16% sodium tartrate, 1% SDS
B 4% CuSO4.5H2O
C Mix 100 parts reagent A with 1 part reagent B to form
alkaline copper reagent.
D Diluted Folin-Ciocalteu 2 N phenol reagent, 1:1 (v/v)
with deionized water

Bradford Solution
Coomassie Brillant Blue G-250.......100 mg
95% EtOH...............................50 ml
85% Phosphoric Acid(w/v)...........100 ml
Distilled Water .........................up to 1 liter

Sample buffer (loading buffer): 62.6 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% $-
mercaptoethanol, & 0.0125% bromophenol blue. Dilute the sample at least 1:4 with sample buffer.

Separating gel solution (12.83%) and stacking gel (4%): Tris-HCl, TEMED, SDS,
acrylamide/bisacrylamide, ammonium persulfate. pH of stacking gel is lower than the pH of the
separating gel.

Electrophoresis Running Buffer, pH 8.3: 25 mM Trisma, 250 mM glycine, 0.1% SDS in 4 liters distilled
water.

Coomassie Brilliant Blue Stain R250

Destaining Solution: 10% acetic acid, 35% methanol
Staining Dye: 45% methanol, 9% acetic acid, 0.25% Coomassie Blue R250

Note: Students are responsible for knowning the purpose of each solution. Students should have a basic
understanding of chemicals frequently used in biochemistry, for example, sodium phosphate buffer.
 35

APPENDIX

PIPETMAN OPERATION
In your lab, you have available three different pipetmen depending on the lab. If you look at the top of
the plunger it states the size of the pipetman. P20 measures accurately from 2 :l to 20 :l. P200
measures accurately from 20 :l to 200 :l. P1000 measures accurately from 100 :l to 1000 :l. Never
turn the pipetman above the maximum volume; 20 :l for P20, 200 :l for P200, and 1000 :l for P1000
as this breaks the pipetman. The scale on the pipettor is read different for each type - refer to Figure 5
for an example of how to read the scale.

(Excerpted from Gilson pipetman operation manual.)

1. Setting the volume: The required volume is set on the digital volumeter by turning the knurled
adjustment ring (Figure 8-2A). When the volumetric setting is increased, it is necessary to go
about 1/3 of a turn above the desired setting and then come back to the exact value. When the
volumetric setting is decreased the desired value may be selected directly. The volumeter display
is read from top to bottom in :l for P20 and P200 and ml for P1000 (Figure 8-2).

2. Place a disposable tip on the shaft of the Pipetman. Press on firmly with a slight twisting motion
to ensure an airtight seal. Depress the push-button to the first positive stop (Fig. 8-3A). While
holding the Pipetman vertical, immerse the tip 2-4 mm into the sample liquid. Release the push-
button slowly to draw up the sample (Fig. 8-3B). Wait 1 to 2 seconds, then withdraw the tip
from the sample.

3. To dispense the sample, place the tip end at a 10-45o angle against the inside wall of the vessel
and depress the push-button SMOOTHLY to the first stop (Fig 8-3C). Wait 1 to 2 seconds and
then depress the push-button completely to expel any residual liquid (Fig. 8-3D). With the push-
button fully depressed, carefully withdraw the Pipetman, sliding the tip along the inside wall of
the tube. Release the push-button. Remove the used tip by depressing the tip ejector button
(Figure 8-1F).
 36

Figure 8: Gilson pipetman operation.

1-A, push-button; 1-B, moulded hand grip; 1-C, shaft; 1-D, built-in ejector; 1-E, tip; 1-F, ejector
button; 2-A, knurled adjustment ring; 3-A, 3-B, 3-C, and 3-D as discussed in operation of push-
button.
 37

OPERATION OF FLOOR MODEL CENTRIFUGES

Note: If procedure varies depending on centrifuge manufacturer a step by step operation procedure is
usually located on or nearby the centrifuge or the teaching assistant will help you.

HITACHI HIGH SPEED HIMAC REFRIGERATED CENTRIFUGE

• to select or change settings the CHECK button must first be pressed (light on). The light stays on
for 16 sec. When the light is off you can no longer select, change setting or carry out any
operation, just press check button again and continue.
• When the centrifuge is turned on and the CHECK button is not pressed. The centrifuge displays
real time parameters.

OPERATION
Centrifuge tubes should be balanced by scale by adding or removing appropriate solution from one of
the tubes.

1. Turn power switch on. The indicators on the control panel are illuminated. The door lock is
released.
2. Open door. If required set the rotor gently in position and close door. Turn the rotor lightly by
hand to check that the rotor is correctly set. Remove the rotor lid and place balanced tubes
opposite each other in rotor. You cannot run the centrifuge with an odd number of tubes.
SCREW ON LID.
3. Call up memory code number or enter parameters.
Call up pre-programmed memory code number: Press CHECK button, MEMORY button,
memory code number, and CALL button. Each memory code number consists of a specified set
of operation parameter (see sheet on centrifuge cover). See below for a list of operation
parameters and how to set and store operation parameters.
OR
Real time operation (enter original parameters): see setting of operation parameters below.
4. After the parameters are set make sure the check light is still on. If not, press the CHECK
button.
5. Press the START button. The rotor starts running. The start lamp begins flashing. The timer
starts to count down.
6. The timer counts down to zero or press the STOP button. The rotor begins to decelerate. The
stop light begins flashing.
7. The rotor stops. The stop light stops flashing. A buzzer sound occurs. The door lock is released.
8. Unscrew rotor lid and remove tubes. If required, use tweezers to help remove tubes. Wipe out
rotor if spills occur. DO NOT SCREW ON THE LID just place on top of the rotor.
9. Close centrifuge lid and turn off power.

PARAMETERS
ROTORS NUMBER: SMALL (maximum volume 40 ml) RPR20-2 = ROTOR #7
LARGE (maximum volume 450 - 500 ml) RPR9-2 = ROTOR #13
TEMPERATURE: 4 to 20 oC
SPEED: Rotor number 7 (small) - maximum speed 18,000 rpm
Rotor number 13 (large) - maximum speed 8,000 rpm
Example: for 3,520 rpm, press 3 . 5 2
TIME 0 to 99 min 59 sec, FREE
Example: for 5 min and 30 sec, press 5 . 3 0
ACCEL. Higher the value the faster the acceleration - 9 is good for basic centrifugation.
DECEL. Higher the value the faster the deceleration - 7 is good for basic centrifugation.
Example: for loose pellet or phase separation the deceleration number should be
decreased to 3.

SETTING OF OPERATION PARAMETERS

1. Press CHECK button, CHECK button lights up. Parameter for preceding operation are displayed.
The LED light goes off after 16 sec, it is only possible to set parameters with the CHECK button
light on. Press CHECK again if light goes out.
2. Press TEMP button. Position where setting is to be made will flash. Using the ten number key
 38

pad, key in desired value for parameter setting. Data will be displayed in the above setting
position. Press SET button. The flashing stops and selected operation parameter is set.
Note: if your require 4oC, set at 8oC as the centrifuge often goes below the requested setting.
Your sample may freeze if spinning for greater than 5 min at a high speed.
3. Repeat step 2 for each remaining parameter...SPEED, TIME, ROTOR NO., ACCEL., &
DECEL. Remember to press the CHECK button again if the CHECK light goes out.

STORING OF OPERATION PARAMETERS (Storing of operation parameters is optional.)

4. After setting desired parameters as above, press CHECK if light has gone out.
5. Press MEMORY, memory code number (one that is available) and the RECORD button. There
are nine available memory program code numbers. See chart on centrifuge lid for memory
programs that already exist.

SPECTRONIC 20D OPERATION

The spectronic 20D is a single beam spectrophotometer. The wavelength range is 340 nm to 600 nm
with a nominal spectral slit width of 20 nm that is constant over the wavelength range. The wavelength
accuracy is 2.5 nm. The spectronic 20D is supplied with 1/2 inch test tubes.

SAMPLE MEASUREMENT: Absorbance

1. Remove dust cover. Turn on Power Switch clockwise. Allow the spectrophotometer to warm
up for 15 min.

2. Set the required wavelength with the Wavelength Control Knob.

3. Set the display mode to TRANSMISSION by pressing the MODE CONTROL KEY until the
LED beside TRANSMISSION is lit.

4. The sample compartment should be empty and closed. Adjust the display to 0.0%T with the
Zero Control Knob (same as power switch).

5. Fill a spec 20D 1/2 inch test tube with blank solution. The tube should be at least 1/2 full. Wipe
the test tube with tissue to ensure no liquid drops, dust or fingerprints. Place the test tube in
Sample Compartment and align the guide mark on the test tube with the guide mark at the
front of the sample compartment. Press test tube firmly into sample compartment and close lid.

6. Press the MODE CONTROL KEY until the LED beside ABSORBANCE is lit. Adjust the
display to 0.0A with the Transmission/Absorbance Control Knob. Remove the test tube from
the sample compartment.

7. Put test tube containing sample(s) in Sample Compartment and close lid. Read absorbance
directly from display.

8. When all measurements are complete, turn off the spec 20D and replace dust cover. Thoroughly
rinse all spec 20D test tubes with distilled water, and place test tubes in spec 20D rack upside
down.

COMMENTS
1. Using the same test tube for blank and all samples should minimize error. Although this is not
usually required for experiments carried out in undergraduate labs.

2. Keep all solutions free of bubbles.

3. The display must be reset to 100%T or 0.0A every time the wavelength is changed.
 39

SWIFT II WAVESCAN OPERATION WINDOWS95 VERSION

(SWIFT computer software attached to Ultrospec 4000 spectrophotometer)

1. Turn on ultraspec 4000. Allow the blinking orange light to turn green before connecting
software package. Cannot open the sample chamber when the orange or red light is blinking.
Warm up time is approximately two to four minutes. Chamber lid remains closed except to add
or remove cuvette from cuvette holder.

2. Double click on INSTRUMENT CONTROL icon. This connects the ultraspec 4000 to the
computer. Turn on the deuterium lamp by clicking on left box - turns blue when the lamp is on.
The tungsten lamp is on all the time (red block). Reduce view.

3. Double click on WAVESCAN icon.

4. Select NEW from the pull down FILE menu.

Under parameters,
-set start and stop wavelengths: 225 nm to 350 nm,
-reference requirements (default) [reference before first samp
- number of samples, change to sample 1 of 2.
-Under details, write title, for example group number, section, operator - student names
-Under display, set minimun and maximum absorbance: 0 to 1-3, leave at default as autoscale
selected in run options. The maximum absorption is 3.0.
Under run options, set post run; save, select overlay and autoscale.

5. Set up 3 quartz cuvettes: standard (absolute ethanol), student oxidized sample, student reduced
sample. Put all samples in cuvette, blue holder (reference, first), then oxidized and reduced.

6. Click running man icon.

Computer prompts for reference.
Click on okay. Computer generates a wavelength scan in an overlay screen.

7. To write on graph (for example oxidized or reduced, peak at 275 nm) select the text button
located right side of screen. Using the mouse locate 275 nm or line and press the right button
and drag to where you want to put the message. Menu comes up that allows you to write or just
click enter and the peak height and nm location will be printed.
8. Select FILE, print.
 40

SAMPLE FINAL LAB EXAM:

Microbiology 60.346 Membrane and Cellular Biochemistry

WRITE IN PEN ONLY

CONCISELY ANSWER ALL QUESTIONS. ANSWERS ACCEPTABLE IN POINT FORM.

1. Briefly answer each of the following questions.

2 a) What is the theoretical bases for choosing 20% diisopropyl ether in chloroform as the thin
layer chromatography solvent for ubiquinone?

4 b) Name each major procedure in the isolation and purification of ubiquinone from beef heart.
What is the main function of each procedure? Do not give procedure details of each step.

1 c) What compound is added to the modified Lowry reagent that allows measurement of red
blood cell membrane protein isolated by the procedure used in your 60.346 lab?

1 d) What is the major chemical component difference between 5PS buffer and PBS buffers?
Explain this chemical component difference relating to red blood cell membrane isolation.

2 e) What is the principle of protein determination by the Bradford method?

1 f) Centrifugation steps for the isolation of red blood cells membrane are performed at 4oC.
Explain why.

2 g) You are asked to determine the molecular weight of a purified protein. The protein consists
of 3 different polypeptides; two of the polypeptides are found in duplicates. Explain how would
you determine the molecule weight of this protein. ONLY present a completely labelled
schematic data that demonstrates the experiment you performed to determine molecular weight.
State conclusion.

1 h) How do you calculate the Rf value of a protein band after SDS polyacrylamide gel
electrophoresis?

3 i) Theoretically a standard curve of log molecular weight vs Rf value of protein band for SDS
polyacrylamide gel electrophoresis is a linear plot. However, experimentally the plot is often not
linear for the higher molecular weight proteins. Explain relationship between theoretical and
experimental results relevant to principle of SDS polyacrylamide gel electrophoresis.

2 j) Discuss the function of the polyacrylamide gel electrophoresis buffer during SDS
polyacrylamide electrophoresis. Include a diagram illustrating the juxtaposition of the
polyacrylamide gel and electrophoresis buffer.

CONTINUED ON PAGE 2....

 41

SAMPLE FINAL LAB EXAM CONTINUED:

Microbiology 60.346 Membrane and Cellular Biochemistry

5 2. Explain the function of each of the following components used in your lab.

a) condenser (isolation and quantitation of ubiquinone from bovine heart)

b) absolute ethanol (isolation and quantitation of ubiquinone from bovine heart)
c) acetic acid and methanol (at all steps of coomaissie blue stain procedure)
d) 2% Na2CO3 and 0.4% NaOH in Lowry Reagent
e) 5% $-mercaptoethanol in polyacrylamide sample buffer

3 3. How do you accurately prepare each of the following solutions for use in the membrane lab?

a) 500 ml 20% diisopropyl ether in chloroform (volume/volume)

b) 1 liter 50 mM NaCl (NaCl formula weight of 58.4)
c) 100 ml Lowry reagent A (2% Na2CO3, 0.4% NaOH, 0.16% sodium tartarate, and 1% SDS)
All reagents are percent weight/volume.

4 4. a) Determine the concentration of ubiquinone (moles/g) in beef heart using the following
experimental data.
i) mass of beef heart sample is 24.0 g
ii) crude ubiquinone sample volume prior to spotting on TLC is 250 :l
iii) crude ubiquinone sample volume spotted on TLC plate is 22 :l.
iv) ubiquinone sample volume collected from TLC elution step is 1.8 ml
v) TLC eluted sample diluted 4 fold before measurement in spectrophotometer
vi) spectrophotometric results for measurement of 1 ml TLC eluted sample
nm Absorbance oxidized Absorbance reduced
250 0.05 0.04
255 0.07 0.06
260 0.12 0.08
265 0.20 0.09
270 0.28 0.09
275 0.24 0.09
300 0.09 0.02
350 0.02 0.02
2 b) Determine the protein concentration (mg/ml) of a membrane sample using the following data.
i) slope of standard curve is 0.08 :g-1 plotting optical density at 590 nm vs :g protein
ii) sample volume measured 10 :l
iii) each tube measured in spectrophotometer had 5 ml Bradford reagent added
iv) spectronic 20D average reading is 0.152 O.D. at 590 nm
___
33

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