JSU Student Symposium 2020
Testing Different Blocking Buffers and Concentration Effects on Signal Strength by Western Blotting Against Cytochrome C



Faculty Mentor

Roger Sauterer, Biology


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Houston Cole Library, 11th Floor | 11:15-11:25 a.m.


Western Blotting is a commonly used process in which specific proteins are detected in a sample of tissue or extract by using primary antibodies that specifically bind to the protein and secondary antibodies which bind to the primary antibody. Blocking is the process of exposing a Western blot to a protein or other solution that sticks to the blot matrix and prevents non-specific binding of antibodies. Different blocking buffers give different results depending on the protein being analyzed, with some stripping off the protein while others enhancing the resulting signal. In these experiments, we tested a variety of blocking buffers at different concentrations, as well as combinations of blocking buffers to determine which buffers gave the most substantial signal with little to no background with Western blotting against cytochrome C. Blocking is a very important step during the Western blotting process. As stated previously, it prevents the nonspecific binding of the antibody to the membrane, which is the leading cause of high background in the Western Blot. The high background leads to inaccurate analysis of the proteins within which is what these experiments were aimed to avoid. All experiments used 5% nonfat dry milk as a control. Trial blocking buffers included Milk at concentrations ranging from 1% to 5%, BSA ranging from 1% to 5%, Hemoglobin also ranging from 1% to 5%, Gelatin at 3%, and, lastly, PVP/PEG ranging from 1% to 4% each. Other combinations included PVP/PEG and combinations of different proteins. Since PVP/PEG combinations provided the best signal with Western Blotting against histones, we expect similar results with Western blotting against cytochrome c.


student presentations, student posters, blocking buffers, proteins


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Molecular Biology

Presentation Information

Sharp, J. and Harris, S. (2020, 13 February). Testing different blocking buffers and concentration effects on signal strength by western blotting against Cytochrome C. Paper presented at the 2020 JSU Student Symposium, Jacksonville State University, Jacksonville, AL.

Testing Different Blocking Buffers and Concentration Effects on Signal Strength by Western Blotting Against Cytochrome C