Blocking Buffers and Their Effects on Mammalian Histone and Cytochrome C

Mammalian histones are highly alkaline proteins found in the nuclei that organize DNA into chromosomes and regulate transcription. Cytochrome c is a heme protein that is found between the inner and outer mitochondrial membranes and functions to transfer electrons of the respiratory chain. We are investigating the interactions between histones and mitochondria and cytochrome C and mitochondria by using cell fractionation and Western Blotting to identify the histones and cytochrome C. The blocking step in the process coats the membrane with proteins or other molecules to reduce non-specific binding of the antibodies. Although 5% nonfat dry milk is widely used as a blocking buffer, we are testing different blocking buffers, as different blocking buffers yield different results, enhancing the signal or reducing it, depending on the specific antibodies used, and the optimal blocking buffer for a given antibody must be determined by experimentation . We use 5% nonfat dry milk (BLOTTO) 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. Since antibodies to histone H3 and cytochrome C 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 to the histone and cytochrome C. Our preliminary results indicated that PVP/PEG combinations provided the strongest signal for both histone and cytochrome C, while gelatin tends to strip the proteins completely off of the blot and reduce the signal. Non-protein blocking buffers in general worked better than protein-based blocking buffers.https://digitalcommons.jsu.edu/ce_jsustudentsymp_2021/1001/thumbnail.jp