I'm trying to detect the change in the level of some proteins after treatment of the cells with a drug. In order to be sure that the low protein level is not, because I loaded less protein, I have to detect the loading control protein GADPH. I load to he gel 20 µg protein.
Hi,
if your protein of interest and GAPDH don't have the same molecular weight, you can detect both proteins simultaneously. In case they do have similar MWs and you can get access to a fluorochrome detection system, you could detect your protein of interest with one fluorochrome and GAPDH with another fluorochrome. You could also try a combination of fluorescence and ECL. Just be aware that in the case of fluorescence detection and PVDF membranes you use low auto-fluorescent membranes. If none of the above options are feasible you can always detect your protein of interest, strip the membrane and reprobe for GAPDH.
Good luck!
As an alternative, if your primary antibodies are raised in different species...then your secondaries will not cross-react with antibodies targeting your various proteins of interest (Secondary A binds Primary A, but Secondary B does not bind Primary A). This would eliminate the requirement to strip your blot and instead you can peroxidase treat your membrane. The peroxidase treatment will essentially "kill" the previously bound HRP-conjugated secondary antibody. From there, you can simply reprobe with Primary B followed by Secondary B. As a feel good, you could always apply ECL to your blot following the peroxidase treatment...it should result in NO signal.
Thanks Bert and Peter for your suggestions, I tried to do mild stripping for the membrane but it did not work and it did not eliminate the first bound antibody so I tried the harsh stripping method and I found that it works well but in the same time I am losing amount of the proteins that bound to the membrane. I will try the flourescently
Dear Fardous,
This stripping protocol using 6M guanidinium hydrochloride has worked the best for me in the past:
Yee-Guide Yeung and E. Richard Stanley. "A solution for stripping antibodies from PVDF immunoblots for multiple reprobing" Anal Biochem. 2009 June 1; 389(1): 89–91.
I've also known people who use a 8M GnHCl with 1mM DTT dissolved in water as their stripping solution, which has also worked well for me before.
Dear Mark,
Many thanks for your interesting paper but I am using nitrocellulose
and I think I will switch to PVDF one
Thanks Janaina,
I tried to cut the membrane but it was destroyed, is there any special type of scalpel that i can use?
Fardous, I would use a new or nearly new razor blade or scalpel to make sure you get a clean cut.
I did want to add that I will only chop blots when I am certain that my antibody is specific only for the protein I am interested in; it's good practice to test your antibody against the whole blot the first couple of times.
Dear Peter Pioli, can you explain more your idea of using two primary antibodies raised in different species?
cutting the membrane works well in case of the proteins have different molecular weights but if they are close to each other it did not work with me. so I am planning to use two primary antibodies raised in different species. it will be nice if you send for me the exact protocol to do that.
Dear Thomas, I tried your potocol for stripping and it work very well.
I would like to thank you for such protocol I did not loss too much protein when I used it.
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