B02B01029

A3

105S3 BIOTECHNOLOGY CORE TECHNIQUES

Protein experiment report

Abstract
This report is for protein part of 105S3 BCT experiment course. This part
contains 6 sections, which are protein extraction, purification, gel filtration, SDS-
PAGE electrophoresis, western blot and immunostaining. The main target of this
experiment is to purify and identify the expressed GUS protein, and finally calculate
its possible molecular weight. This report includes the documentation of experiment
results, the material and method we used and the discussion based on what we did
and observed.

Introduction
GUS is a gene encoding for -glucuronidase, which is a member of the
glycosidase family of enzymes that catalyze breakdown of complex carbohydrates.
-glucuronidase is synthesized as an about 80 kDa monomer and exist as a
honotetramer which has a size of 332 kDa. The product of GUS gene can turn X-gluc
into blue compound, and this character make the gene an ideal reporter gene which
is commonly used in the field of biological research.
Gel filtration is a method for protein separation by molecular size. By beads
with micro pores, the mothed creates extra length of path for smaller molecular as
them flowing down the column and then separated with the larger ones. In this
experiment, we used Sephacryl S-300 resin (MW range 10-1500 kDa for globular
proteins) to pack our column.
Affinity chromatography separate target proteins by specific binding between
wanted molecules and the ligands linked on the beads. Ni-NTA agarose beads was
used in this experiment to recognize and purified the His-tag attached GUS protein
from out protein sample extracted from E.coli.
To detect whether our desired GUS protein is in the sample, we used anti-
GUS first antibody and HRP-linked secondary antibody and then stained the detected
proteins with DAB substrate solution. The HRP added on the secondary antibody can
turn DAB into colored compound, which can be used for signal presentation,
observation and quantification.
Results
We prepared our protein samples by precipitation in 70% ammonium sulfate
environment after repeatedly freezing and thawing the bacterial cells for lysis. The
supernate we got from centrifuge after lysis is XT, and the ammonium sulfate purified
XT is called TP. TP then went through dialysis processing to get rid of the salts in it,
and the desalted TP was named DI. DI was used in affinity chromatography for
further purification of GUS protein, and the product, AF, would be applied in gel
filtration for a molecular weight measurement of GUS protein in the latter
experiment.
The XT, TP, DI and AF were collected and analyzed together by SDS-PAGE
acrylamide gel electrophoresis and immunostaining. We made two SDS-PAGE gel
electrophoresis with different protein molecular weight markers. One was LMW-SDS
marker and the other was BLUltra Prestained marker. The gels then went through
CBR gel staining and western blot respectively. The results are showed at fig.1,2. In
the result of commasive blue stain, we can see that the more sample we loaded, the
thicker and clearer the bands are. Theoretically, the samples treated with more
purification process wouldve presented a clearer bands pattern, and the AF ones
shouldve had only single band. But obviously the result didnt fit our prediction well.
The western blot result still showed lots of bands which with smaller molecular size
than the GUS one.

Fig. 1

Fig.1 The commasive blue stain of SDS-PAGE gel electrophoresis. The first and last 3 lanes
are loaded by XT and AF with different loading amount of sample. The fourth and fifth lanes
are for TP and DI.
Fig.2 The western blot immunostaining result. The samples are the same as the fig.1.
 Fig. 2

The 26 tubes of affinity chromatography products were also tested by enzyme

activity assay. The measurement of enzyme activity and protein concentration was
conduct by 415nm and 595nm absorbance respectively with Microplate reader. We
use serial diluted BSA solution as standard to calculate the actual concentration of
protein in our samples. The results are showed at fig.3,4. We can see the peaks of
595nm and 415 nm appeared at the almost same position, which indicate a
successful
isolation of GUS
protein from the
DI sample by the
affinity column.

Fig.3
The affinity
chromatography
result. The blue
line is 595nm

Fig. 4 absorbance and

the orange one is
for 415nm.
Fig.4
The serial dilution
standard curve.
2=0.9711
 To roughly calculate the molecular weight of GUS protein, we applied our AF
sample and other 5 different protein markers together to a gel filtration column. The
markers are used for the establishment of a molecular weight standard. The 5
markers are thyroglobulin (MW=670000), globulin (MW=158000), ovalbumin
(MW=44000), myoglobin (MW=17000) and B12. With enzyme activity assay analysis,

Volume MW logMW
thyroglobulin 100 670000 5.826075
globulin 120 158000 5.198657
ovalburin 135 44000 4.643453
myoglobin 155 17000 4.230449
B12 1355 3.131939
GUS 117.5 184587 5.2662
Gel filtration Fig. 6
415nm enzyme activity 595nm protein concentration

2.5

1.5

0.5

0
32.5
2.5

17.5

47.5

62.5

77.5

92.5

107.5

122.5

137.5
145
152.5

167.5

182.5
10

25

40

55

70

85

100

115

130

160

175

Fig.5 The regression equation generated

we can realize when the GUS protein
from the 4 peaks of markers and their
flowed out the gel filtration column.
molecular weight.
The result is showed at fig.5,6.
Fig.6 The result of affinity column. The blue
The 4 peaks of markers
line is 415nm absorbance, and the orange
appeared at the volumes of 100, 120,
one is for 595nm.
135 and 155 mL, and the peak of GUS
was located at the position of 117.5 mL. By a simple calculation, we made a rough
estimation of 184.5 kDa on the molecular weight of GUS protein, which is actually
about 200 kDa.

Discussion
The weak effect of affinity chromatography on the commasive blue staining
result. We cant see the different between patterns of the samples before and after
the affinity column purification. The impurity bands still existed in the AF samples.
Because we can still find some good results from other groups experiment record,
this was probably not the material problem. There might be several possible causes
of the inefficient purification like bad column packing, adding too much sample at
the same time and the disturbance of the upper surface. We dont know which one
was actually happened, or maybe we just messed up a little at the every steps.
Incorrect molecular weight. The molecular weight of GUS protein indicated
by the commasive blue stain result was between 45 kDa and 66 kDa, but its actually
80 kDa. There are several possible reasons for this situation. One is the degradation
of the proteins during the experiment. According to out western blot result, our GUS
protein sample remained a correct size of 80kDa, so the problem didnt come from
the samples itself. Its also no possibility of that we used an incorrect protein
molecular weight marker because it really did have 6 bands on the same positions as
the tutorial. I think that the flawed acrylamide gel casting was the most likely reason
in this case. It might cause some problems during electrophoresis and make a speed
difference between ladder and samples.
The non-specific binding bands in the western blot result. We can see that
except for the ones with GUS protein, there are still lots of other bands in the
membranes with the smaller molecular weights. They are not the normal residual
proteins due to bad purification. They were recognize by the antibodies and stained
by DAB solution. It means that they were or carried some fragments from GUS
protein. There are two reasons for the appearance of them. One is that the
incomplete blocking made the membrane able to bind the antibodies not specifically.
The others is the degradation of the GUS protein, which might produce many smaller
pieces. These little fragments then were recognized and stained, and finally formed
the bands we see. I think the second explanation is more possible, because if it was a
incomplete blocking, the whole membrane shouldve cover by the stain. Obviously it
didnt happen and these bands all appeared below the main band of GUS protein. So
the degradation seems to be the most likely cause.