Problem when reprobing after stripping a membrane during Western blot
dan_su at hotmail.com
Thu Mar 18 14:48:25 EST 2004
in this condition you certainly will lose a lot of your protein on the
membrane, to my experience about 1/3 to 1/2 of the proteins will lose in
such stripping conditions, I would suggest you to use more gentle stripping
buffers, lower temperature and shorter time, expose your film longer.
<hugues.chanteux at facm.ucl.ac.be> wrote in message
news:20040318093951.12401.qmail at ww02.hostica.com...
> 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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