Hi,
I am going to do Percoll gradient centrifugation for subcellular fractionation. Before I start I would like to know whether I need to adjust the PH of percoll to 7.4. I use percoll from Sigma (PH 8.5-9.5).
And after centrifugation, can I directly use the fractions for SDS PAGE and WB?
Thank You
Anu
11/26/18
Dear CJ,
The need to adjust the pH will likely depend on the nature of your sample. Are you trying to isolate and characterize one or more proteins or enzymes? If so, then you need to be concerned about the pH stability of these materials. If their activity or functionality are pH-sensitive, then the safe approach would be to adjust the pH to 7.4, assuming that is the pH at which they are stable/functional.
After isolation, you should be able to go directly to SDS-PAGE. I have never worked w/ Percoll, but it is a colloidal silica suspension. It might not hurt to try a centrifugation step to remove the particles. If they can't be removed this way, I would expect them to remain at the top (or "origin") of your gel, and therefore not be a problem. One way to find out would be to add some Percoll to one of your protein standards and run this in a separate "lane" on your gel to see how it behaves.
At the electrophoresis step, the pH of your sample should not be a problem, as the SDS-PAGE buffer should be sufficient to override the sample pH and allow your proteins to migrate through the gel.
I hope this information helps you.
Bill Colonna Center for Crops Utilization Research, Iowa State University, Ames, Iowa, USA [email protected]
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