Protein buffers such as PBST, which is used in western blotting, are normally adjusted to pH 7.4. When I try to find why, I find some information about optimal pKa for protein stability. Im not sure I really understand this, because it seems as through regardless of protein - most laboratories just use PBST with a pH of 7.4.
I am a bit curious about this, and have a few questions
- Why is pH 7.4 set as the standard for western blotting?
- For an optimal western blots, should the pH always be above the pI of the protein?(since 7.4 seems a bit high)
- Should the pH of a buffer be at the pI of a protein for extractions before SDS-pAGE, and while washing NC-membranes after blotting?
- How should one calculate the optimal pH, or evaluate/think about this when working with a protein?