Hi all, I am using M-PER Mammalian Protein Extraction Lysis Reagent to lyse my cells for western blot. The protocol given was to spin the cells down at 14,000g for 15mins and collect the supernatant, but I accidentally spin it down at 14,000 rpm = 18,000g instead. Will spinning at such a high speed degrade my protein further, thus decreasing the quality of my samples and blots?
It is highly unlikely that excessive g force will affect your protein especially in the range of g that you mention. I strongly doubt that the authors of your protocol optimized the g force and set it at 14 000 g and not 18 000 g. Most likely, this was the force available with their centrifuge and they found that 15 min was sufficient to pellet cellular debris.
I agree with Dr Pierre, probably it will not affect your protein.
In the Sigma website there is a chart showing the RPM to G convertion based on the radius of the rotor and a calculator too.
sigmaaldrich.com/US/en/support/calculators-and-apps/g-force-calculator
Depends and should follow protocol if G and if rpm, both can be converted based on the type of centrifuge machine in the lab.
I fully agree with the first answer and would like to add that it's not possible to degrade a protein by centrifugation alone. Note that some protocols require high speed centrifugation above 100.000 g (e.g. isolation of membrane fraction) which doesn't result in protein degradation (other than enzymatic proteolysis).
What about the extraction of RNA Samples, if i wash it with WB1 @ 14000Rpm b'coz usually i do it @8000Rpm for 1 minute
and what about the Elution Buffer usePankaj KumarHüseyin BesirPankaj KumarPankaj KumarPankaj Kumar
pre heated or not for the same b'coz i don't use pre heated Elution Buffer Maddie YeoAntonio CasalinhoPankaj Kumar
Pankaj Kumar Hüseyin Besir alright, thank you for the responses. It's true, my protein came out perfectly fine in my western
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