Hello
I have cultured cells and I want to extract proteins before using the PNGase for deglycosylation and SDS PAGE. The protocol from the provider for the PNGase is pretty simple, but what buffer would you use to harvest your proteins? Is RIPA suitable (I've seen some protocols with it but have a doubt as it already contains SDS and so on). Thanks you for your help.
If you are working with a membrane protein, my suggestion would be to purify the membranes containing the protein, instead of dissolving them. You can treat the membranes with PNGase F to deglycosylate the protein, then run SDS-PAGE. This avoids the presence of a detergent during the digestion. You can use the same buffer to purify the membranes that is recommended for the PNGase F digestion.
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