How to solubilize membrane proteins of human postmortem brain tissue for western blot analysis?

#1

Hello,

I would greatly appreciate any advice on preparing tissue for western blot with human postmortem tissue. I can get bands for my proteins, but they are variable and it seems to be most variable at the upper molecular weights (membrane bound proteins) and not the lower molecular weight soluble proteins so I think it is a solubilization issue. I have tried RIPA and RIPA with urea (although urea prevents me from using an enzyme I need) with sonication and got similar results. I also tried 2% SDS in tris buffer and boiling, but it made my proteins precipitate and not migrate into the gel. Does anyone have tips for solubilizing membrane proteins from human tissue? Thanks!

#2

Hi @Cheryl,
Thanks for posting! The research we do at the Allen Institute for Brain Science has not leveraged Western blotting in protocols using human brain tissue, so we don’t have any specific advice to offer on this, but hopefully someone from the community might be able to weigh in.
We have encountered a few protocols in the literature that might be useful to refer to, as a starting point, though we have not tested any of them ourselves.

  • Springer Protocols: Reis-de-Oliveira, et al, “A Complete Proteomic Workflow to Study Brain-Related Disorders via Postmortem Tissue.”
  • Segerstrom, et al, “Minimizing Postsampling Degradation of Peptides by a Thermal Benchtop Tissue Stabilization Method.”
  • Ferrer, et al, “Brain Protein Preservation Largely Depends on the Postmortem
    Storage Temperature”
  • Blair, et al., “Individual Case Analysis of Postmortem Interval Time on Brain Tissue Preservation.”

Good luck with your experiments!

#3

Hello,

Thank you so much for the response and useful resources. It is very much appreciated! At least I am not alone in solubilization troubles with the postmortem
tissue. I will keep trying!

Best,

Cheryl