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Western problem : no signal - could that be linked to pH? (Jun/19/2007 )

Hi all,
I'm trying to do a western, and can't see anything, not even background. I used a standart HRP labelled secondary antobody with pierce ECL kit.
Transfer seems to be ok, as I can see proteins on my membrane, antibodies are supposed to work (I used them successfully not that long ago), same thing with the ECL kit. One thing that I noticed is that the pH of the TBS I used this time was around 10 instead of around 7.5. Could that explain the fact that I can't see anything (I do have a positive control on my gel)?? I could not find any information linking pH to western problems...
Thank you so much for your help!

-zera-

the optimum pH for HRP activity is 6.0-6.5 activity at 7.5 is about 84% of the max. So I think your pH may be the problem... see this page from Sigma (I googled "pH for HRP activity" it was the first listing.)

http://www.sigmaaldrich.com/Area_of_Intere...se_Enzymes.html

-beccaf22-

QUOTE (zera @ Jun 19 2007, 07:13 PM)
Hi all,
I'm trying to do a western, and can't see anything, not even background. I used a standart HRP labelled secondary antobody with pierce ECL kit.
Transfer seems to be ok, as I can see proteins on my membrane, antibodies are supposed to work (I used them successfully not that long ago), same thing with the ECL kit. One thing that I noticed is that the pH of the TBS I used this time was around 10 instead of around 7.5. Could that explain the fact that I can't see anything (I do have a positive control on my gel)?? I could not find any information linking pH to western problems...
Thank you so much for your help!


for each Ab used, optimum ionic strength and pH must be optimized; most start with the standard PBS or TTBS protocol or follow the recommendations of the manufacturer; pH 10 would probably not support any specific antigen-antibody interaction

-The Bearer-