Wenzhou-Kean University

Immunology Lab Report 5

Western Blot Analysis

Zhonghao Chen

Partner: Changyi Sun

BIO 4316 Lab W01

5/6/2021

Abstract
 Western blotting is a very sensitive and convenient method to identify and screen

the proteins in immunological experiments. In this lab, students are asked to use

bovine serum albumin (BSA) to perform western blotting and analyze the results.

Although in this experiment, we did not get good results, there are still useful

experiences and lessons that can be learned from this experiment.

Introduction

Western blotting, also called protein immunoblotting, is a scientific technique by

which an individual protein is visualized amid thousands of other proteins in a sample

(Roy et al.). This method is commonly incorporated with other techniques like gel

electrophoresis and enzyme-linked immunosorbent assay (ELISA) which can help

scientists do detailed proteomic studies (Kompoliti and Leonard). For example, in

2009, SNEED et al. used western blot analysis to compare the structural proteins of

avian infections bronchitis virus that assisted them to understand the pathways and

mechanisms of the virus’ variation.

The progress of western blotting can be mainly divided into three steps that are

running a denaturing polyacrylamide gel, the direct transfer of protein from the

polyacrylamide gel to a charged nitrocellulose membrane, and immunological

detection. First, in the running of a denaturing polyacrylamide gel step, sodium

dodecyl sulfate (SDS) and 2-mercaptoethanol should be used to denature the protein.

This ensures that the complex configurations of the proteins can be destroyed which

assists to accurately resolve the sizes of proteins. Then, in the next step, the protein

should be transferred from the gel to the charged nitrocellulose membrane. The reason
is that the membrane can bind proteins with a high affinity and the membrane are

much more pliable than gel. Finally, for immunological detection, the principle of this

step is similar to ELISA. The specific primary antibody will bind to the absorbed

protein antigen, and a secondary antibody liked an enzyme against the primary

antibody is then applied. In the end, the membrane will be incubated with a solution

contains substrates for the enzyme that can show the position of the bands.

In this lab, students are asked to use bovine serum albumin to perform Western

blotting. Our group hypothesizes that we can observe the marker on the membrane

after immunodetection and through the marker, we can speculate the molecular

weight of the bovine serum albumin (BSA). We cannot observe the band of negative

control solution, but we can observe the band of BSA high concentration solution and

BSA low concentration solution on the membrane. Besides, the band of BSA high

concentration solution will darker than the BSA low concentration solution.

Methods

Reagents preparation The reagents prepared in this lab can mainly be divided into

three kinds. The first kind is the regents for gel electrophoresis that includes pre-

stained protein standard markers, negative control solution, BSA high concentration

solution, and BSA low concentration solution. The second kind of reagents is

immunochemical and blotting reagents such as anti-BSA protein antibody, secondary

antibody conjugate, hydrogen peroxide (stabilized), peroxide co-substrate,

10×blocking buffer, powdered milk, and 10×PBS. The third kind of reagents is other

chemical reagents that include a 10×Tris-Glycine-SDS buffer (chamber buffer),

10×Tris-Glycine powdered buffer (transfer buffer), nitrocellulose membrane, filter

paper, and large filter paper (wick).

The preparation of the polyacrylamide gels and chamber The polyacrylamide gels

should be set as Figure 1. shows. Remove the cassette, comb carefully, and the gels

should be inserted into the electrophoresis chamber. The electrophoresis buffer should

be added into the chamber and the buffer should cover the top of the shorter plate.

Rinse each well by squirting electrophoresis buffer into the wells.

Figure 1. The set of the polyacrylamide gel electrophoresis (Millipore Sigma).

The loading of protein samples 20 μl of pre-stained protein standard marker,

negative control solution, BSA high concentration solution, and BSA low

concentration solution should be loaded into the appropriate lane of the gel (because
the gels are shared by groups).

The running of the gels Once all samples have been loaded, carefully place the cover

onto the electrode terminals and connect the electrical leads to the power supply. Set

the voltage of the power that is approximately 100V to apply and perform

electrophoresis. After the electrophoresis is finished, carefully remove the gel cassette

from the electrophoresis apparatus and blot off the excess buffer with a paper towel.

The front plate of the gel cassette should be removed and the gel should be placed into

the transfer buffer to soak for 10 minutes.

The Western Blot procedure Pre-soak wick, blotting paper, and nitrocellulose

should be put in transfer buffer for 5~ 10 minutes. Place a large gel plate on the top of

a container approximately 16×9×4 cm (length×wide×deepth). The transfer buffer

should be added to the tray and the presoaked wick onto the gel plate such that ends

are submerged in 2 cm of transfer buffer as Figure 2. shows.

Flip the gel and place the gel flat on top of the wick. Smooth over the top of the

gel to remove air bubbles. Place nitrocellulose membranes on the top of the group’s

respective gel lanes and two pieces of blotting paper should be put on the top of the

membrane. Finally, a 6cm stack of paper towels should be put on the top of blotting

paper and about 1 kg weight should be placed on the top of the stack to complete

assembly as Figure 2. shows.

Figure 2. The side view of the blot transfer assembly (Millipore Sigma).

Immunodetection Dismantle the stack above the membrane and carefully move the

membrane from the gel with forceps or a spatula. A labeled small sealable plastic bag

with 10 ml blocking buffer should be prepared. The membrane should be submerged

into the solution and occasionally agitate for 5 minutes. Discard the blocking buffer

and 10 ml of primary antibody solution should be added into the plastic bad. Incubate

the membrane for one hour at room temperature on a shaking platform and discard the

primary antibody solution. The membrane should be washed for 5 minutes in 10 ml

blocking buffer. Discard the blocking buffer and repeat wash. 10ml of secondary

antibody solution should be added to the plastic bag. Incubate the membrane for one

hour at room temperature on a shaking platform and discard the primary antibody

solution. The membrane should be washed for 5 minutes in 10 ml PBS. Discard the

blocking buffer and repeat wash. The membrane should be washed with water and

then air dry on a paper towel. Compare the size of the samples containing the various

concentrations relative to the protein standard markers.

Results
 In this lab, we only can observe the markers on the membrane as Figure 3 (a).

shows. We also referred to the results got by Junyi Wang and Qiwen Tian as Figure 3

(b). shows. Because they used another batch of membranes provide by Green, the

markers on the membrane cannot be clearly observed.

Figure 3. Part (a) of this figure is the results got by Changyi Sun and Zhonghao Chen.

Part (b) 0f this figure is the results got by JunyiWang and Qiwen Tian.

Discussion

From the results, we can conclude that our experiment is failed. The reason is that

there are no bands for the BSA high concentration solution and BSA low

concentration solution that are different from the expected results. This also means

that the results we got reject our hypothesis. About this phenomenon, our group

proposes two possible reasons that can explain it.

Above all, in the immunodetection progress, the primary antibody or secondary

antibody we used may be denatured. The reason is that all of the groups failed in this

experiment. Because of the denaturation of primary antibody or secondary antibody,

they cannot bind the antigens effectively and may be washed away by buffers. This

may result in that there is no enzyme that can catalyze the substrates and we cannot

observe the bands of BSA high concentration solution and BSA low concentration

solution in our results. The other reason may be the insufficient soaking tine of

substrates, or the substrate may be decomposed by other enzymes encountering during

the experiment. In a word, the loss efficacy of substrates can also result in the

disappearance of bands.

However, this experiment still makes sense because it displays the important

procedures and the meanings of them. First, the electrophoretically fractionated

proteins should be transferred to a membrane for immunological detection. One of the

reasons is that the membrane is conducive to the performance of immunodetection. At

the same time, the membrane is stronger and pliable than gels. Secondly, before

transferred proteins can be detected using antibodies, the membrane must be

incubated with a blocking buffer to block the unoccupied binding surfaces on the

membrane. Thirdly, there are two primary advantages of western blotting that are

sensitivity and specificity when comparing to the total protein stain. Finally, a positive

control lysate is from a cell line or tissue sample that is known to express the protein

of interest. This control can ensure that there were no issues in the western blotting

protocol. Whereas, a negative control can determining whether non-specific binding

(false-positive result) has occurred in the western blotting procedure.

Taking together, in this experiment, students learn the procedures of western

blotting and understand the mechanisms behind them. Although the whole experiment
is failed, there is still much useful information that can be learned from this

experiment. For future experiments, we need to find useful ways to detect the activity

of antibodies and substrates before using them. This will be one of the research

emphasis in further research.

 Works Cited

Millipore Sigma. Northern and Southern Blot Protocols & Introduction. 6 May. 2021,

www.sigmaaldrich.com/technical-documents/articles/biology/southern-and-

northern-blotting.html#reference

Kompoliti, Katie, and Leonard Verhagen. Encyclopedia of movement disorders. Vol.

1. Academic Press, 2010.

Roy, Jyoti, et al. "Small RNA proteome as disease biomarker: An incognito treasure

of clinical utility." AGO-Driven Non-Coding RNAs. Academic Press, 2019. 101-

136.

SNEED, LOYD W., et al. "Comparisons of the structural proteins of avian infectious

bronchitis virus as determined by Western blot analysis." Viral immunology 2.3

(1989): 221-227.