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
>>http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0
