Roger Sauterer, Biology
Download (69.6 MB)
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
This video is the property of Jacksonville State University and is intended for non-commercial use. Video and images may be copied for personal use, research, teaching or any "fair use" as defined by copyright law. Users are asked to acknowledge Jacksonville State University. For more information, please contact email@example.com.
Sharp, Jerod and Harris, Shelby, "Testing Different Blocking Buffers and Concentration Effects on Signal Strength by Western Blotting Against Cytochrome C" (2020). JSU Student Symposium 2020. 26.