Hi All,
I am quantifying a specific protein from cells that have been pelleted and resuspended in 90% FBS/10% DMSO (not by design of this particular experiment). My goal is to calculate a ratio of specific protein to overall protein from these samples. I have the specific protein quantification handled, but I am using a BCA assay to calculate total protein. The problem is...FBS is full of protein. The samples are currently at -80, so we're talking at least 1 freeze/thaw cycle before anything can happen.
Can I spin these samples down to re-pellet my cells, thereby removing the excess protein from FBS? Would there be any lingering consequences from their initial suspension in FBS?
Much thanks in advance for any advice you may have,
Ariel
Your cells are actually suspended in a typical freezing mix, designed to protect them during freezing, and allowing further culture. Simply thaw them (fast), resuspend them in culture media without protein and centrifuge (at gentle speed, just enough for the cells to pelletize) , resuspend at least a couple of times (you may measure supernatant protein in order to monitor effiency of the washing). When you are sure that they are washed, lyse and measure. If in doubt whether they survive, use a viability marker to stain the cell suspension.
If you froze and thawed your cells, they are broken and you won't be able to separate the released proteins from the serum. You can still determine total membrane proteins by spinning the sample at 100,000 check, washing the pellet, and re suspending before solubilizibg and doing BCA. So you are kinda screwed unless you start over, and wash the cells to remove FBS before quantifying,
Your cells are actually suspended in a typical freezing mix, designed to protect them during freezing, and allowing further culture. Simply thaw them (fast), resuspend them in culture media without protein and centrifuge (at gentle speed, just enough for the cells to pelletize) , resuspend at least a couple of times (you may measure supernatant protein in order to monitor effiency of the washing). When you are sure that they are washed, lyse and measure. If in doubt whether they survive, use a viability marker to stain the cell suspension.
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