I lysed my cells in lysis buffer (Thiourea, urea, DTT, Triton X-100, tris and protease inhibitor) for 2DE analysis. Unfortunately, we had to switch to in solution digestion instead of in-gel digestion. For LCMS, I was told that thiourea and detergents cannot be present in my samples. So now I am looking for way to remove detergent and thiourea completely from my samples. Can in-gel digestion with SDS PAGE or protein precipitation using TCA/acetone help to remove all these unwanted components? I did try filtering using 3K MWCO filter but it still had traces of detergent. Please help me out with your experiences. Thank you
Hey there,
acetone precipitation of peptides sounds like a good way to remove detergents which should be soluble in this solvent. This method even works with membrane proteins to remove detergent micelles.
For larger portions (mL range) of detergent-containing solutions, BioBeads are useful. These particles adsorb all the detergent molecules from your sample and can be removed by centrifugation or filtration.
For small portions (µL range) of detergent-containing solutions that have to be prepared for mass spectrometry, ZipTips are particularly useful. They can be used with usual pipettes and work like small-scale C18 columns. You could elute your peptides with an intermediate amount of acetonitrile while the more hydrophobic detergents remain on the tip matrix.
Good luck!
Removal of detergents from protein solutions is one of the difficult task indeed, particularly if your target proteins have high surface hydrophobicity.
Did you try Aqueous two-phase extraction (ATPE)?
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