Harika Divi

Pls refer to the following article which introduce publication guidelines for a good reliable Western Blot:

DOI: 10.1016/j.ab.2020.113608

Hello Harika Divi

You can ensure the specificity and sensitivity of Western Blot results if you follow these measures.

To carry out a successful Western Blot, the quality of the primary antibody is a critical factor. Therefore, validation of the antibody will be important to avoid inaccurate results. Validation would include determining the optimal antibody concentration for the protein of interest. After validating that the antibody specifically recognizes the target protein it is important to determine the linear dynamic range for the target protein using the validated antibody against the samples being investigated. Different antibodies show different linear dynamic ranges, especially at high total protein levels. It is critical that the western blot be carried out within the linear range of the antibody being utilized. This can be easily carried out by doing Western Blots using different sample dilutions.

The specificity of antibodies can readily be determined using positive and negative controls. The best positive controls would be purified proteins or lysates which overexpress the target protein, while the best negative controls would be tissues from knock-out animal tissues or cell lysates.

Another factor to consider are the housekeeping proteins which are excellent normalization references when validated for the tissue or cell being investigated. But recent findings suggest that these housekeeping proteins are substandard loading controls under many conditions. Reasons include the presence of numerous poor-quality antibodies to each housekeeping protein, the relatively high expression of housekeeping proteins in many tissues and cells, and the change in expression of housekeeping proteins in some tissues under certain experimental conditions.

Both the protein of interest and the housekeeping protein need to be within the linear detection range for their expression levels to be accurately determined. Housekeeping proteins are high abundance proteins and therefore are often overloaded, particularly when large amounts of total protein are loaded to detect low abundance target proteins, so that housekeeping protein expression levels cannot be quantified accurately. Another overlooked aspect of housekeeping proteins is that they are highly post-translationally modified, which can also potentially affect quantification depending on the epitope of the antibody utilized.

Total protein quantification by Coomassie blue, Ponceau S, and the Stain-Free method has been shown to have advantages over housekeeping proteins for normalization of Western Blots mainly since this normalization does not depend on expression of a single protein.

There are some more measures which you could follow to ensure that your Western Blot results are reliable. You may have to refer to the article attached below.

Article The necessity of and strategies for improving confidence in ...

Best.

Go read your notes for help with your homework. Reported.

The specificity and sensitivity can be checked by using the internal controls like beta actin or GAPDH before starting the actual experiment.