Problem when reprobing after stripping a membrane during Western blot

hugues.chanteux at facm.ucl.ac.be hugues.chanteux at facm.ucl.ac.be
Thu Mar 18 04:39:51 EST 2004


Dear all,

I have some problem with reprobing my membrane after stripping.

After ECL detection, I directly put my nitrocellulose membrane in PBS.  After rincing the ECL, The membrane are incubated with stripping buffer (TRIS 62,5 mM-SDS 2% - Mercaptoethanol 100 mM - pH 6,7) 30 min at 50°C under gentle agitation.  After, membrane are washed several times with PBS until no odour of mercaptoethanol can be smelt.  Then, membrane are reblocked and reprobed with primary antibody overnight at 4°C.

The first probing works very well (detection of  the phosphorylated protein).  It is when I want to detect the non-phosphorylated protein on the same blot (after stripping) that I get no signal.  There is no problem with the antibody because when I first try to detect the non-phosphorylated protein, it works.

If somebody could help me, it would be grateful.

Thanks in advance

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