EXPERIMENT No.

3
ISOLATION AND CHARACTERIZATION OF PROTEINS

A. Isolation of Ovoglobulin and Ovalbumin from egg white

Measure the
Separate the
volume of egg Stir gently until
egg white from Add 3.60 g of
white using a pre- no further
the yolk of one NaCl powder
weighed graduated precipitation of
medium-sized for every 10 mL
cylinder. ovoglobulin is
egg. Discard of the sample.
Determine weight observed.
the yolk.
of sample.

Filter the residue Add 2-3 drops of Treansfer the Filter using
using cheese 0.200 M acetic residue to pre- cheesecloth. Do
cloth. Transfer acid to the weighed filter not iclude
precipitate to filtrate. Heat to paper and air undissolved salt
pre-weighed filter boiloing until dry sample. powder. DO NOT
paper and air dry ovalbumin Weigh dried THROW
the sample. precipitates. sample. FILTRATE.

B. Quantification of Protein using Biuret Method

Prepare 2.00 mL of
Separate the egg Measure the the
each albumin
white from the volume of the egg using
standard in separate
yolk of one graduated cylinder.
test tubes with
medium-sized DIvide the sample
concentration of 5,
egg. Discard the evenly to 250 mL
10, 15, and 25 mg/ml
yolk. beakers.
of distilled water.

Read absorbance using

Add 2.00 mL of Transfer 2.00 mL
a UV-Vis
Biuret reagent of egg white into
spectrophotometer. If
and 1.00 mL of
the absorbance is two separate test
0.200 M NaOH to
exceeding 1.00, dilute
each prepared tubes as unk.
with distilled water samples. Label
tubes. Incubate
until absorbance properly.
for 30 mins.
reading is below 1.00.
 C. Qualitative Test for Protein

Dilute egg white

sample with equal Transfer 25 mL of the TEST TUBE 1
volume of 0.200 M egg white solution to 2.00 mL of ninhhydrin
phosphate buffer. four separate test solution, heat for 5
Filter through tubes. Label properly. mins in water bath.
cheesecloth.

TEST TUBE 4
5 drops of glycoxylic TEST TUBE 2
acid, tilt tube slightly TEST TUBE 3
5 drops of
and carefully add 5 drops of Millon's
concentrated nitric
2.00 mL of reagent, heat for 5
acid, heat for 5 mins
concentrated sulfuric mins inn water bath.
in water bath.
acidto form two
layers.

C. denaturation of Protein

TEST TUBE 1
0.200 M HCl drop by drop until cahnge
occurs.

TEST TUBE 2
0.200 M NaOH drop by drop until cahnge
occurs.

TEST TUBE 3
0.200 M lead acetate drop by drop until
cahnge occurs.

TEST TUBE 4
0.200 M urea drop by drop until cahnge
occurs.
TEST TUBE 5
0.200 M tannic acid drop by drop until chage
occurs.

TEST TUBE 6
70% ethanol drop by drop until chage occurs.

TEST TUBE 7
heat in water bath until change occurs

TEST TUBE 8
agitate vigorously until change occurs