Resuspending protein mixture after over-centrifuging - (Sep/05/2021 )

Hello!! I was hoping someone might be able to help me with an issue I have during extraction for sarcomere proteins from mice heart tissue.

I had mice heart tissue samples from the right ventricle. I'm trying to isolate protein from the sarcomere for a western blot.

I started by Homogenizing the samples in SRB with Triton, proteases, and phosphotease. Then I was supposed to centrifuge at 200G for one minute at 4 degrees Celsius. Instead, I made a mistake and centrifuged at 20,000G. I ended up with a big solid precipitate in the bottom.

I continued my normal protocol hopping it'll work. I isolated the liquid from the precipitate. I think the liquid should be the cytosol and it's proteins. Then, I added SRB to the precipitate and centrifuged again. Then used added urea and centrifuged for the last time at 17,000G for 10 minutes at room temperature.

At the end, is still had the same looking solid precipitate. I isolated the soluble liquid and did a BCA. Turns out there is no protein in the soluble liquid I isolated. I saved the precipitate hoping I'd find a way to troubleshoot and fix the mistake. Any ideas?

Unfortunately, likely no way to recover it, as it can be really hard to re-solubilize proteins once they have precipitated. It is likely that the initial big solid precipitate is where your proteins are, not in the supernatant after this step.

Depending a bit on what you want to do, you could try heating it in a buffered 1% SDS solution (e.g. denaturing loading dye), but even this may not work.