Roger Sauterer, Biology
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Houston Cole Library, 11th Floor | 4:00-4:10 p.m.
Mammalian histones are highly alkaline proteins found in the nuclei that organize DNA into chromosomes and regulate transcription. We are investigating the interactions between histones and mitochondria by using cell fractionation and Western Blotting to identify the histones. The blocking step in the process coats the membrane with proteins or other molecules to reduce non-specific binding of the antibodies. We use 5% nonfat dry milk as a control for all experiments, it is essential to use this variation of milk because it reduces background noise and helps produce good, clear bands. Although 5% nonfat dry milk is widely used as a blocking buffer, we are testing different blocking buffers to see if the signal strength increases or completely strips proteins off of the blot, the conclusion of which blocking buffer works the best will be determined by the protein being examined. Since antibodies to histone H2A and H2B are low-affinity and give a weak signal, we are therefore testing different blocking buffers, such as milk concentrations ranging from 1 to 5%, BSA ranging from 1 to 5%, and hemoglobin ranging from 1 to 5%, as well as gelatin at 3%, and PVP or PEG individually ranging from 1 to 4% and different combinations of the aforementioned blocking buffers to determine which blocking buffer gives the best detection signal. Our preliminary results indicated that RVR/REG combinations provided the strongest signal, while gelatin tends to strip the proteins completely off of the blot and reduce the signal.
student presentations, student posters, histones, blocking buffers
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Tavis, Peighton and Harris, Shelby, "Blocking Buffers and Their Effects on Mammalian Histones" (2020). JSU Student Symposium 2020. 30.