Hi everyone, I’m working with samples that have a significant insoluble fraction after lysis and centrifugation. I would like to run the pellet (insoluble) part on a Western blot, either because the protein of interest is membrane-associated, forms aggregates, or is otherwise not in the supernatant.

Could anyone please share a reliable protocol or tips for:

• Solubilizing the pellet without degrading the protein?
• Suitable buffers (e.g., urea, SDS, Triton X-100)?
• Sonication or heating steps?
• Any precautions to preserve epitope integrity for antibody recognition?
• Compatibility of solubilized pellet samples with standard SDS-PAGE and blotting?

Any references, tips, or reagent suggestions are most welcome. Thanks in advance!