Western blot - Stripping and Reprobing - (Mar/04/2005 )
I've probed the same blots for different antibodies, tubulin after using a phospho antibody. No problems.
but after using this srtripping protocol I got weak signal when I probed the membrane with another primary antibody for another protein (CHOP), is there any recommendations??
thanks in advance
i use another buffer, 50mM tris pH6.8, 100mM b-mercapto and 2% SDS. Leave on for 30 mins at 50C and then wash off twice with TBS-T
I've probed the same blots for different antibodies, tubulin after using a phospho antibody. No problems.
but after using this srtripping protocol I got weak signal when I probed the membrane with another primary antibody for another protein (CHOP), is there any recommendations??
thanks in advance
Hi!
If you are familiar with the size of your proteins you could easily cut your membrane in pieces and stain each with seperate antibodies. I usually do it this way because it saves you a lot of time and there is no need to strip the membrane. I don´t really trust signals of a stripped membrane especially when the results are weird to interprete.
Cheers
Hi!
We used the same protocol as Patirl and it does not work very well...mostly the bands are weak or results are completely confused...for example: I have a strong bands in cytosolic fraction and weak in membrane fraction, but when I strip the membrane the results were reverse...then i had to use a new membrane anyway to make sure it was a mistake...and it realy was! So now we mostly don't use stripping in our lab, becase u can't believe the results from it.
Kofi
I use 100 mM 2-Mer, 2%SDS, 62.5 mM Tris-Cl pH 6.8 incubated at 50 C 30 min...but the signal protein 18 kDa really week ,but for the B-Actin band it's nice signal ...
Can I strip PVDF membrane today and store in TBST 4C and then reprobe next day??
Normally, How to manage the membrane after took image and when going to strip/reprobe ??
I think I will add more concentration antibody ...
before stripped,I use 1-ab polyclinal 1:1000 and 2-ab IgG HRP 1:2000... they are nice signal..
Does anyone have some experience to advice?
thank 
Can I strip PVDF membrane today and store in TBST 4C and then reprobe next day??
Normally, How to manage the membrane after took image and when going to strip/reprobe ??
I think I will add more concentration antibody ...
before stripped,I use 1-ab polyclinal 1:1000 and 2-ab IgG HRP 1:2000... they are nice signal..
Does anyone have some experience to advice?
thank
My advice is : first probe the lowest signal, i.e. the 18 kDa, strip, and then probe B-actin.
Yes you can strip today, and block overnight and reprobe tomorrow.
if block over night, will my specific protein be blocked ??
Thank for your advice
I've probed the same blots for different antibodies, tubulin after using a phospho antibody. No problems.
I use this stripping buffer too
sometimes it works well though signal get's a bit weaker
and sometimes though conditions are the same my whole membrane goes blank, grrrhhh
I'm so fed up with westerns at the moment
Yes you can strip today, and block overnight and reprobe tomorrow.
if block over night, will my specific protein be blocked ??
Thank for your advice
blocking involves low affinity interactions, antibodies have high affinity interactions with antigen, and will easily compete with the blocking agent.
I don't think you can block too much.
Hi anyone strip using ponceau S solution before? I have tried using that and it worked for me...