27 Apr 2012
How to get better phospho-blots?
If you are trying to detect phosphorylation of a protein using a phospho-specific antibody and get an unsatisfactory weak signal, what usually helps is supplementing 1 uM of microcystin-LR to the lysis buffer just prior to cell lysis.
26 Apr 2012
Strong western blot stripping protocol (home-made):
Strong Stripping Buffer: 1% SDS, 63mM Tris-HCl, pH 6.8, 0.5% Tween 20, 0.7% beta-mercaptoethanol (BME).
For 1L: 31.5 ml of 2M Tris-HCl stock, 100 ml of 10% SDS stock, 5 ml of Tween 20, 863.5 ml dH2O. Add BME fresh: 70 ul per 10 ml of the buffer.
1) Rinse the membranes with TBST (5 min, while shaking).
2) Incubate the membranes for 5 min at room temp in 0.2M NaOH.
3) Incubate the membranes for 30-60 min at room temp in Stripping Buffer.
4) Rinse
the membranes with TBST (3 times, 5 min each, while shaking).
5) Optional: block as usual.
6) Continue with primary antibody incubations.
EDTA is a divalent cation chelator. There are many enzymes that would not function without their cofactors and in many cases these cofactors are divalent cations such as Mg++. Mn++, Ca++ and Zn++. If a lysis buffer is supplemented with 1-10 mM EDTA, then metalloproteases, nucleases, phosphatases, kinases and several other types of enzymes will be inactive. This is done to preserve the post-translational modification state of the proteins at the moment of cell lysis or to prevent nucleis acids from being degraded.
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