Student : Novi Setiati

Student Id : 111821606
Topic : Western Blot

A. Introduction
Developed in the late 1970s and early 1980s, Western blotting is a popular analytical
technique for identifying one or more particular proteins in a complicated protein mixture.
Initially, gel electrophoresis was used to separate proteins by molecular weight, followed by
electrophoretic transfer to and immobilization of the proteins on a solid nitrocellulose membrane
support, probing of the membrane with antibodies specific for the protein of interest, and detection
of the bound antibody using radio-labeled staphylococcal Protein A followed by autoradiography
for visualization (Azure biosystem, 2019). Western blotting is a fundamental technique in cell
and molecular biology. It is also known as immunoblotting or protein blotting. It is used to detect
the presence of a specific protein in a complicated mixture recovered from cells. The Western
blotting procedure relies on three key elements, that is size separation of protein mixtures using
gel electrophoresis, efficient transfer of separated proteins to a solid support, and specific
detection of a target protein by appropriately matched antibodies. When the target protein is
recognized, it appears as a band on a blotting membrane, X-ray film, or imaging device (Guidance
et al., 2009). Western blotting is one of the most frequent laboratory methods because it can be
done quickly using simple equipment and affordable materials. The results obtained are likewise
simple to interpret, one-of-a-kind, and unambiguous. As a result, it is regularly utilized in research
and clinical settings, either alone or in conjunction with other immunoassays. The figure below
provides an overview of the technique:

Figure 1: Overview of Western Blotting. Separation of protein mixtures by electrophoresis,

transfer to a blotting membrane, and detection of target protein, which only becomes visible in
the final stage as a band similar to that shown in lane 3. Lane 1: Prestained molecular weight
standards. Lanes 2&3: Protein mixtures.
B. The steps of Western Blot
1. Gel Electrophoresis
In the first step of a Western blot, proteins are physically separated from one another across a
gel matrix in a process called gel electrophoresis.

Figure 2: a protein sample is combined with a loading buffer, put onto the gel, and then the
gel/buffer system is exposed to an electrical current. Under the experimental conditions, the
negatively charged proteins migrate through the gel towards the positive electrode.
2. Transfer to Membrane
Proteins are transported to a solid membrane support for further stages after electrophoretic
separation across the gel. Efficient transfer is determined by the membrane employed, the type
of transfer apparatus used, and the composition of the transfer buffer (Biji T. Kurien, 2020).

Figure 3 : Semi-dry Western Blot places the electrodes directly in touch with the
gel/nitrocellulose membrane sandwich, resulting in a fast, efficient transfer. To remove
electrophoresis buffer salts and detergents, the polyacrylamide gels must be equilibrated in
transfer buffer, and the nitrocellulose membranes and filter sheets must be pre-wetted, but that
is all the buffer necessary. When setting up the transfer, it is vital to exclude excess moisture
and air bubbles caught in the filter papers and membrane; often, a pipet rolled over the surface
will take care of this; otherwise, the set-up for this operation is relatively straightforward.
Preparation for semi-dry Westren Blot transfer that is make 1X transfer buffer freshly. A
recipe for 1x transfer buffer (48mM Tris base, 39 glycine, 20% methanol) for 1.0 (Sino
Biological, 2023):

For 1.0 5.76 g Tris Base

L 2.95 g glycine
200 ml methanol
Add 1 L ddw

3. Membrane Blocking
While Western blotting antibodies normally have a high affinity for a specific protein, they
also have a non-specific and low affinity for the Western blotting membrane. When the blot
is photographed, the non-specific binding can cause a large background signal, lowering
detection sensitivity. After transfer, the membrane is incubated in a blocking solution to
reduce background signal as much as feasible. The blocking solution acts by attaching to non-
specific antibody binding sites on the membrane, occluding antibody binding (Li-Cor, n.d.).
4. Membrane Incubation with Antibody
After blocking, the membrane is ready for antibody probing, and unbound antibody is rinsed
away. The type of detection method used, the quality and type of antibodies available, the
number of antigens to be detected, the type of enzyme or tag used for detection, and the
incubation and wash conditions are all factors that influence probing and washing (Butler et
al., 2019).
5. Antibody Detection
The membrane is now ready for antibody probing, and unbound antibody is washed away. All
of these elements influence probing and washing, including the type of detection technology
utilized, the quality and type of antibodies available, the number of antigens to be identified,
the type of enzyme or tag used for detection, and the incubation and wash conditions (Cino et
al., 2020).
C. Troubleshooting
While Western blotting is a reasonably simple and uncomplicated technique, it may not
always produce the expected results. When this happens, it is advantageous to be able to swiftly
isolate the potential causes and devise an appropriate solution by troubleshooting the experiment.
The remainder of the troubleshooting concerns can be divided into three categories: no bands,
dim bands, and signal on Western blots that interferes with bands. As a result, probable causes
and solutions have been arranged in this manner, as well as potential problem sources, such as
antibody, antigen, method, or buffer-related issues where relevant (Guidance et al., 2009).
1. Unusual or Unexpected Bands
Difference seen Possible Cause Action/Solution
Band(s) at lower molecular  Target protein has been  Use a fresh sample which
weight than expected cleaved or digested has been kept on ice
 Splice variants exist  Add fresh protease
 Another protein bearing inhibitors to the lysis
the same/similar epitope buffer
has been detected by  Try alternate antibody
antibody
Band(s) at slightly higher Protein may be glycosylated  Use enzymes to remove
molecular weight than or otherwise modified at one suspected modification
expected, and may be blurred or more amino acid residues returning molecular weight
closer to expected
 Check amino acid
sequence and literature
Band(s) at significantly Dimers, multimers, or  Add fresh DTT or bME to
higher molecular weight than proteinprotein interactions samples and reheat before
expected may be occurring because repeating experiment
samples have not been  Prepare new samples with
fresh loading buffer
Multiple bands at various Primary antibody  Use an affinity-purified
molecular weights concentration may be too primary antibody
high, or there is a cross-  Optimize primary
reactivity with similar antibody concentration
epitopes on other proteins  Try another antibody
 Check antibody specificity
with blocking peptide
2. No Bands
Source of Problem Possible Cause Action/Solution
Antibody related Inappropriate secondary  Retrace steps to check
antibody used compatibility between
primary and secondary
antibodies
 Reprobe with correct
secondary or strip blot and
reprobe if necessary
 Repeat experiment with the
correct antibody
combination
Wrong concentration of  Increase the antibody
antibody or low affinity to the concentration 2-4 fold
target protein
 higher than initially
recommended
 Increase length of
incubation
 Test/optimize antibody on
dot blots
 Try another antibody
Antigen related Antigen not expressed in the  Use another source of
source material target protein
Not enough antigen loaded on  Check concentration of
the gel sample
 Increase the amount of
source material
 Immunoprecipitate,
fractionate, or concentrate
the sample

3. Faint Bands or Weak Signal

Source of Problem Possible Cause Action/Solution
Antibody related Primary or secondary  Repeat using higher
antibody concentrations were concentration of antibody
too low  Optimize antibody
concentration with dot
blots
Low antigen-antibody binding  Reduce the number of
affinity wash steps to a minimum
 Increase the antibody
concentration 2-4 fold
higher than the
recommended starting
dilution
Antigen related Insufficient sample loaded on  Check concentration of
the gel sample
 Increase the amount of
source material
 Immunoprecipitate,
fractionate, or concentrate
sample
Blot has been stripped and  Redo blot since antigen
reprobed may have been stripped off
or damaged by stripping
process
Buffer related Non-fat dry milk may mask  Decrease % of milk in the
some antigens blocking and antibody
solutions
 Try alternate blocking
solution
 Reference

Azure biosystem. (2019). Western Blotting Guidebook. 4.

https://www.bu.edu/picf/files/2019/05/Azure-Western-Blotting-Guidebook.pdf
Biji T. Kurien, R. H. S. (2020). Western Blotting.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304528/
Butler, T. A. J., Paul, J. W., Chan, E. C., Smith, R., & Tolosa, J. M. (2019). Misleading westerns:
Common quantification mistakes in western blot densitometry and proposed corrective
measures. BioMed Research International, 2019. https://doi.org/10.1155/2019/5214821
Cino, A., Domingos, E., Gueorguiev, B., Haraj, M., D’Esposito, A., Pirek, C., Arsimoles, D., Bulla,
M., Sassi, A., & Guilhen, C. (2020). The AJ521 antibody detects the human CD1b protein by
western blot. Antibody Reports, 3(1), e122. https://doi.org/10.24450/journals/abrep.2020.e122
Guidance, T., Analysis, D., & Moore, B. C. (2009). Introduction to Western Blotting Western
Blotting. MorphoSys UK Ltd.
Li-Cor. (n.d.). Odyssey TM Western Blotting Protocols. Odyssey.
Sino Biological. (2023). Semi-dry Western Blot Transfer.
https://www.sinobiological.com/category/wb-semi-dry-transfer