Hi, I am trying to extract cytosol-enriched and nuclear-enriched fractions from cultured cells. For the cytoplasmic extraction, I am using a hypotonic lysis buffer that contains DTT (http://cshprotocols.cshlp.org/content/2019/7/pdb.rec102004.short). This seems to work fine (I have checked with western blotting using GADPH as a cytosol marker) but I'm running into problems when I want to measure total protein content using the BCA assay which isn't compatible with DTT. (DTT reacts to produce color that messes with the assay.)
Has anyone tried cytoplasmic extraction using a buffer that doesn't have DTT? For example, a hypotonic buffer that's exactly the same except that I don't add DTT to it?
Thank you!
Dithiothreitol (DTT) is added to the extraction buffer to maintain the reduced state of thiols in proteins in the cytoplasm. You could extract without using a reducing agent if you don't mind oxidation of protein thiols, which could potentially lead to intermolecular disulfide crosslinking, non-native intramolecular disulfide bond formation, and/or oxidation of cysteine side chains to cysteic acid. The importance of these changes would depend upon what you plan to do with the extract.
If your extraction buffer does not contain detergents, then you could use the Bradford assay instead of the BCA assay, since the Bradford assay is not affected by DTT.
Hi amrutha,
DTT half life is 30 min at pH 8.0, upto 2-3 mM shd be fine during the assay
secondly try using TCEP (upto 5mM is compatible) a combination of DTT and l-cysteine ( 2+ 4mM shd be more effective and less interfering) should help the cause
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