Hello,

I have 100 tissue samples (50 cortex and 50 hippocampus) that have been homogenized (sigma RIPA + sigma protease inhibitor (PI)). I have made three supernatant aliquots per sample.

I tried to run my BCA analysis for 39 samples today (as many as would fit onto my 96-well plate in duplicate with 9 standards). In the process my samples had to obviously be thawed and then re-frozen. I have two questions about protein viability throughout the whole process:

1. By the time I am done pipetting the last samples onto the plate (on ice), the first samples I plated have been sitting for let's say 15 minutes. The samples are each diluted in the wells with the same lysis/PI cocktail as they were during homogenization. I wonder if, during this time, my proteins are being damaged because of the temperature increase and how that might affect the PI's 'job'. Is it best to run fewer samples or is this a non-issue?

2. I will have to thaw and re-freeze these samples several times for my westerns. Will this be ok? Does this depend on the proteins I'm looking at? Should I have tried to aliquot the supernatant for as many blots as I will be running?

I appreciate any advice!