Re-Imaging without Stripping - (Aug/05/2010 )
Recently I was attempting to image an experimental blot that had been successfully incubated in primary, secondary, and ECL solutions; however, when I went to image the blot in our Imager, I found that the camera had broke and thus couldn't take any pictures...
Are my blots all wasted? Is it possible to simply wash the ECL substrates off the blot and try again later? Or does the Secondary-HRP get used up? I would really prefer not to strip my nitrocellulose membrane because I'm afraid the process will rip off too much of my experimental protein.
Thanks!
if stored properly (refrigerate wet with buffer, no azide) the hrp should survive.
mdfenko on Fri Aug 6 16:05:08 2010 said:
if stored properly (refrigerate wet with buffer, no azide) the hrp should survive.
For future references, I presume this only works if exposure to ECL reagent was very brief. Thats great to hear though! I called Santa Cruz Biotech about this and everyone said the HRP substrate will get used up the instant it touches ECL.
I think there is a little confusion regarding the HRP versus the ECL. The HRP (horseradish peroxidase) is an enzyme that oxidizes a substrate. This oxidation reaction with chemiluminescent substrates produces light. The HRP enzyme can remain active as long as it is properly stored as previously mentioned (refrigerate in buffer, no azide). Every once in awhile I'll have the enzyme die and then all you need to do is reprobe with the secondary antibody but usually the HRP remains active for days even after extended exposure to ECL. ECL contains the HRP substrate as well as "enhancing" chemicals, usually modified phenols. So, the moment you expose your blot with the HRP-conjugated antibody to the ECL, the substrate begins to be used up but it doesn't destroy the HRP enzyme. If you need to store your blots for awhile (more than a few days), be sure you change the buffer. If bacteria begins to grow in the buffer, the pH will drop and destroy the HRP. But if it does, or if the signal is too weak, just reprobe with the secondary antibody or protein A-HRP.
rkay447 on Fri Aug 6 19:38:59 2010 said:
I think there is a little confusion regarding the HRP versus the ECL. The HRP (horseradish peroxidase) is an enzyme that oxidizes a substrate. This oxidation reaction with chemiluminescent substrates produces light. The HRP enzyme can remain active as long as it is properly stored as previously mentioned (refrigerate in buffer, no azide). Every once in awhile I'll have the enzyme die and then all you need to do is reprobe with the secondary antibody but usually the HRP remains active for days even after extended exposure to ECL. ECL contains the HRP substrate as well as "enhancing" chemicals, usually modified phenols. So, the moment you expose your blot with the HRP-conjugated antibody to the ECL, the substrate begins to be used up but it doesn't destroy the HRP enzyme. If you need to store your blots for awhile (more than a few days), be sure you change the buffer. If bacteria begins to grow in the buffer, the pH will drop and destroy the HRP. But if it does, or if the signal is too weak, just reprobe with the secondary antibody or protein A-HRP.
Oh, wow! Thanks for the great explanation. That's what I suspected, but wasn't too sure about. I guess this means that I can easily image the same blot multiple times over a few days.
You say you just reprobed with more secondary antibodies if you find that your signal is too weak- aren't the secondaries from the first probe still bound to the primaries, but with inactive conjugate HRP? If thats the case, than won't they will get in the way of subsequent secondary antibody incubations?
Also, I was told that the ECL substrates actually binds to the HRP enzyme(according to Santa Cruz, very tightly). Do you think several washes with TBST to remove excess ECL reagents before storage (in TBST) will suffice?
Thanks again!