Hello, I'm trying to analyze the proteome of some tissue by mass spectrometry. After lysing the samples and centrifuging them, sometimes there is quite a bit of fat floating on top of the supernatant.
Even assuming that I have enough material to take only the "clean" part, I wonder: should I? Or should I rather leave it all there and hope that it gets discarded when I precipitate the proteins (e.g. Acetone)?
I'm thinking that I might be losing lipophylic proteins (e.g. membrane transporters) if I remove the part of the supernatant which floats on top. But I also don't want to risk having fat messing around with ...anything. How do you go about it?
Thanks in advance.
I think it is better to work on the material without removing the fat because that may lose you some lipoproteins
What kind of lysis buffer do you use? does it contain any detergent?
For proteomics studies, I would suggest to increase the complexity of samples gradually, in order to get reproducible results. For instance, you can use triton-x114 for the fractionation of hydrophilic and hydrophobic proteins. If you do in-gel, then you can separate them using SDS-PAGE individually and prepare samples for mass spectrometry
thank you both.
it happens with both RIPA buffer (this version: http://cshprotocols.cshlp.org/content/2006/1/pdb.rec10035.full plus protease inhibitors) and 4% SDS buffer.
Erman Kocak thanks for the tip! so you mean loading into the machine only a part of the proteins at a a time? How much protein would you load on the gel to prepare the samples like this?
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