Hi, I performed differential centrifugation: 3,500 rpm for 15 minutes, and the resulting supernatant was further centrifuged at 45,000 rpm for 1 hour to obtain the cytosolic and microsomal fraction. ( I have confirmed with GAPDH). After WB, most of the membrane receptor was in the cytosolic fraction. It is biollogically possible? what could be the reason? I mean, I expected to find it in the membrane fraction since is a transmembrane receptor, and also when is sintetized is anchored in the endocytic vesicles membrane and when is internalized too...Any comments would be appreciated. Thank you!
Hi Erica. I'm not familiar with your method but did you lyse your cells before the centrifugation or did you use some other method to break them? If you lyse them using detergent, your receptor would be released from the membranes, depending on the detergent and concentration used.
Hi Anna, thank you for your answer. Yes, I homogenized the samples using a high-speed homogenizer in lysis buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1% NP40 and 5 mM EDTA supplemented with 1% Triton X-100, 1 mM PMSF, protease inhibitor cocktail, 50 mM NaF, and 1 mM sodium orthovanadate.
So NP40 is a powerful detergent used to break open all membranes within a cell including the nuclear membrane...so all what is anchored to the membranes is released to the cytosol?
Yes both NP40 and Triton X-100 at 1% concentration will solubilize your membrane proteins. You should avoid detergents in your preparation, proper homogenization might be enough so separate your organelles.
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