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?
  • 2
    $\begingroup$ That's the average pH of extracellular fluid and cytoplasm but it can vary. Compartments such as lysosome and mitochondrial matrix would have a different pH. Functional studies with native proteins should employ pH buffers with right pH. During western blots, proteins are denatured and so their isoelectric points/functional pH do not matter. The pH in SDS-PAGE is adjusted according to isoelectric point of glycine which helps in stacking of the proteins. $\endgroup$
    Commented Jul 9, 2016 at 16:28
  • $\begingroup$ Thanks! So basically all you would care about is the pI of glycine, which is why pH 7.4 is the optimal pH for western blotting. $\endgroup$ Commented Jul 10, 2016 at 12:40
  • 1
    $\begingroup$ Well.. it matters for SDS-PAGE.. Not for the blotting step. A specific pH may have differential effect. I have not really thought about that. Good question. I'll look up. $\endgroup$
    Commented Jul 10, 2016 at 18:51
  • $\begingroup$ Thanks again, this is interesting and I think its important to get the basic understanding of how things work. I will add this reflection as a point in my question above! $\endgroup$ Commented Jul 12, 2016 at 12:40

1 Answer 1


The correct pH depends on the properties of the proteins that you are trying to exploit. These are a good deal different between extracting proteins from tissue and performing Western blots.

Extracting proteins means keeping them soluble in aqueous solution. Working at the pI of the protein might be the worst choice as the protein then has no net charge, typically reducing its water solubility. An extreme case would be the highly basic histone proteins with very high pI values but best extracted at low pH.

After proteins have been run on a denaturing gel and transferred to a membrane for Western blotting, they are already denatured and bound strongly to the membrane through hydrophobic interactions little affected by pH except perhaps at extremes. For the blotting, you want to exploit the interaction of epitopes on the protein with the antibodies against them. During antibody production, the interactions between the epitopes and the antibody-specific receptors on the antibody-producing cells occur at normal extracellular body pH, 7.4. Using that pH for the blotting best recapitulates the charge state of both the epitopes and of the antibodies as they existed when cells producing the best antibodies were selected.


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