Western Blot: Technique, Theory, and Trouble Shooting

Western Blot: Technique, Theory, and Trouble Shooting

Western blotting is an important technique used in cell and molecular biology. Researchers can identify specific proteins from a complex mixture of proteins extracted from cells using Western Blot.

A mixture of proteins is separated based on molecular weight through gel electrophoresis. The gels are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labels antibodies specific to the protein of interest. The bound antibodies are then detected by developing the film.
The multi-tag positive loading control protein is used to demonstrate that your Western Blot protocol is efficient and correct and that the antibody recognizes the target protein which may not be present in the experimental samples. Loading such protein into your positive control lane results in a reliably detectable protein sample. It means all the steps of your Western blot functioned adequately, including gel electrophoresis, protein transfer to a blotting membrane, membrane blocking, and antibody labeling. It also gives you greater confidence that the results in the other lanes are real rather than artifactual.

We strongly recommend the use of a positive control protein when setting up a new experiment; this will give you immediate confidence in your Western Blot protocol.

When trying to detect low-abundance proteins, it is especially important to know that your Western blot is functioning as expected. If you detect your protein of interest in the control lane, then an absence of the protein in other lanes is probably directly related to its low abundance rather than a faulty step in the blotting protocol.


  • Demonstrate that your protocol is efficient and correct.
  • Perfect positive control protein for your Western blotting.
  • Save time and resources for all your protein research projects.
  • Give you confidence in your Western blot protocol.
  • Can be used for immuno-precipitation, affinity purification, Western blot and dot blot.

Order this positive loading control protein now: https://lifetein.com/peptide-product/multitag-protein-p-1.html
This paper provides the theoretical explanation of the Western Blotting procedure, troubleshooting tips for common problems: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/
Troubleshooting: (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/)

Even though the procedure for western blot is simple, there could be many unexpected results:
(1) Unusual or unexpected bands
(2) No bands
(3) Faint bands or weak signal
(4) High background on the blot
(5) Patchy or uneven spots on the blot
(6) Non-fat milk or BSA

(1) Unusual or unexpected bands: These can be due to protease degradation, which produces bands at unexpected positions. It is advisable to use a fresh sample that had been kept on ice or alter the antibody. If the protein seems to be in too high of a position, then reheating the sample can help to break the quaternary protein structure. Similarly, blurry bands are often caused by high voltage or air bubbles present during transfer. In this case, it should be ensured that the gel is run at a lower voltage and that the transfer sandwich is prepared properly. Also, changing the running buffer can also help the problem. Nonflat bands can be the result of too fast of travel through the gel, due to low resistance. To fix this, the gel should be optimized to fit the sample. Finally, white (negative) bands on the film are due to too much protein or antibody.

(2) No bands: This is due to many reasons related to the antibody, antigen, or buffer used. If an improper antibody is used, either primary or secondary, the band will not show. Also, the concentration of the antibody should be appropriate as well; if the concentration is too low, the signal may not be visible. It is important to remember that some antibodies are not to be used for western blot. Another reason for no visible bands is the lowest concentration or absence of the antigen. In this case, an antigen from another source can be used to confirm whether the problem lies with the sample or with other elements, such as the antibody. Moreover, prolonged washing can also decrease the signal. Buffers can also contribute to the problem. It should be ensured that buffers like the transfer buffer, TBST, running buffer, and ECL are all new and noncontaminated. If the buffers are contaminated with sodium azide, it can inactivate HRP.

(3) Faint bands or weak signal: It can be caused by a low concentration of antibody or antigen. Increasing exposure time can also help to make the band clearer. Another reason could be nonfat dry milk masking the antigen. In this case, use BSA or decrease the amount of milk used.

(4) High background on the blot: It is often caused by the too high concentration of the antibody, which can bind to PVDF membranes. Another problem could be the buffers, which may be too old. Increasing the washing time can also help to decrease the background. Additionally, too high exposure can also lead to this problem. Therefore, it is advisable to check different exposure times to achieve an optimum time.

(5) Patchy or uneven spots on the blot: Improper transfer usually causes them. If there are air bubbles trapped between the gel and the membrane, it will appear darker on the film. It is also essential to use a shaker for all incubation so that there is no uneven agitation during the incubation. Once again, washing is of utmost importance as well to wash the background. Antibodies binding to the blocking agents can also cause this problem; in this case, another blocking agent should be tried. Filtering the blocking agent can also help to remove some contaminants. Finally, this problem can also be caused by the aggregation of the secondary antibody; in this case, the secondary antibody should be centrifuged and filtered to remove the aggregated.

(6) Which blocking buffer/blocking agent do I use? Non-fat milk or BSA?

Non-Fat Milk


  • cheaper
  • Blocks better than BSA
  • Easy preparation


  • Cannot be used with antibodies which target phosphoproteins
  • Cannot be used with streptavidin-biotin-based systems as milk also contains biotin
  • Lowers the sensitivity of some commercially available anti-His monoclonal antibodies



  • Provides defined results – only one protein means lower risk of cross-reactivity
  • As Albumin is not generally phosphorylated, it can be used with phosphoproteins.


  • More expensive
  • Not recommended for lectin probing as its carbohydrates may increase background