I am trying to isolate native protein from both mouse gut tissue as well as human gut samples. Could anyone suggest a better protocol or lysis buffer recipe for this experiment. I intend to use it for immunoprecipitation later on.
That depends on whether your protein is expressed in the cytosol, the membrane, inside organelles or the interstitial space. Cytosol for example is high K + Mg, interstitial fluid high Na + Ca. The pH is also different in different compartments too, the buffer concentration should be about 50 mM. The Good's buffers were designed to interfere as little as possible with proteins.
In any case, you will need a protease inhibitor cocktail (available commercially), and depending on the sensitivity of your protein some polyol to bring the osmotic pressure to 300 mOsm (glycerol, sucrose, mannitol). Some proteins also need an SH-buffer (DTT, mercaptoethanol) or are most stable when they have a ligand bound to them. For transmembrane proteins you also need detergents to solubilise them out of the membrane.
Thank you so much Dr Engelbert for your answer. I will follow your suggestions accordingly.
Regards,
Alpana
I don't Know if RIPA buffer would be appropiate. Maybe Tris glycine. Maybe suggest it may also depend on the protocol used. You may need RNAse or DNAse
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