Hi all,
I need to detect the phosphorylation status of a brain protein. We use SynPER reagent for preparing synaptosomes from frozen brain samples and include phosphatase and protease inhibitors. Before homogenezation the tissue is thawed on ice and, dependent on the number of samples, may stay on ice for a while (up to 20-30 min). Do you think that this will affect the phosphorylation status of the protein i.e. is phosphorylation that sensitive? In addition, any advice on preparing tissue for phospho-WBs would be much appreciated.
Best wishes,
Tau
Dear Tau,
For common lysates phosphatase inhibitors should protect phospho-protein for the 30 minutes you mention. But when using chunks of tissue it will depend in how accessible are the intracellular phospho-proteins to the inhibitors.
The best way is to process the samples as fast as possible after dissection, but in your case you should test the isolation of synaptosomes and bloting of samples treated vs non treated with the inhibitors in order to assess the differences. Then you can adjust the times and dosage of inhibitors that better fit your purposes.
Good luck!
Thanks a lot Sebastian! I will try your suggestions.
Best wishes,
Tau
The kinetics of dephosphorylation depends on the target protein. If you are talking about neuronal activity markers such as CREB or MAPK there are losing their P marker within seconds even on ice and there is no magic protease inhibitors mix to help you, whatever the vendor says. If you do a time-course always shuffle your samples loading order, boil your sample right after -80C while it is still frozen (or better right after dissection) and add your denaturation buffer directly without thawing the sample.
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