Hi all,
I have been having problems with my blots recently which I cannot explain logically so I have a few questions about the basics of Western blot and hoping your answers can shed some light on my Western blot mysteries:
1. How stable is HRP on secondary antibody? When I don’t get signals on film, I usually try different ECL solutions (using CST SignalFire™ ECL #6883, Amersham ECL RPN2106 and home-made ECL with coumaric acid, luminol and H202). I wash the membranes (2x10 min) with TBST in-between using (different) ECL. Is that a proper method? How do you prepare membranes when you want to repeat the ECL? Can using ECL (different ones) consume activity of HRP?
2. In case of empty film, is there any sense in repeating the incubation in new aliquot of secondary antibody on top of already bound primary-secondary complex on membrane?
3. How stable is the interaction between primary antibody and membrane (PVDF in my case)?
I asked in previous topic if the prolonged storage (more than few days) in TBST buffer (0.1% Tween) can affect primary antibody bound to membrane.
4. How stable is the interaction between primary and secondary antibodies? As in previous question, can prolonged storage in TBST buffer affect complex of primary and secondary antibody?
Thank you in advance for you replies.
1 - You could try more sensitive ECL kits to increase the sensitivity of your reaction, but you need to do again blocking, primary and secondary antibodies incubation. The reason is the absence of signal do not mean the absence of enzymatic reaction but a too weak signal to be detected. With its substrat, the stability of HRP enzyme lower due to the enzymatic reaction.
2 - Do you think your secondary antbody is not good ? You have repeated Freeze/thaw ? If yes, do it. You need to know that HRP enzyme did not "like" sodium azid. But if you have no problem with this tube, no it is not necessary.
3 - The stability of antigen and primary antibody is unpredictable. Only the experience could determine it.
4 - Idem before. It is the same problem. Often the stability is better but....
I have a question : the western blot, without the SDS-PAGE/transfert, take 24H if you do overnight incubation. I do not understand why you want to keep probed membrane for more than this time. You use PVDF membrane, for long storage, you could dry it on wattman paper and keep it at minus 20°C. And between each use, you could do this procedure. One use is all WB steps, including ECL. You just need to do each time the blocking procedure. In case of...
Hi Josip
Those are a lot of question and I can only answer a few that I experienced personally. I have tried using different ECL solutions and in my opinion if I do different ECL exposure back to back the same day, 10 min TBST wash is fine.
However, If Blots are kept in TBST, I prefer to incubate again in secondary Ab, it’s definitely better.
You asked for long term storage, and as far as I know, storing for too long in TBST reduces the Ab bound to epitope.
Thanks
1 - You could try more sensitive ECL kits to increase the sensitivity of your reaction, but you need to do again blocking, primary and secondary antibodies incubation. The reason is the absence of signal do not mean the absence of enzymatic reaction but a too weak signal to be detected. With its substrat, the stability of HRP enzyme lower due to the enzymatic reaction.
2 - Do you think your secondary antbody is not good ? You have repeated Freeze/thaw ? If yes, do it. You need to know that HRP enzyme did not "like" sodium azid. But if you have no problem with this tube, no it is not necessary.
3 - The stability of antigen and primary antibody is unpredictable. Only the experience could determine it.
4 - Idem before. It is the same problem. Often the stability is better but....
I have a question : the western blot, without the SDS-PAGE/transfert, take 24H if you do overnight incubation. I do not understand why you want to keep probed membrane for more than this time. You use PVDF membrane, for long storage, you could dry it on wattman paper and keep it at minus 20°C. And between each use, you could do this procedure. One use is all WB steps, including ECL. You just need to do each time the blocking procedure. In case of...
1. That's probably a sufficient wash, especially since you weren't getting a detectable signal to begin with. The HRP should be fine with that too.
2. Yes, you can definitely re-incubate with your secondary. For very weak signals, I'll sometimes do my secondary overnight at 4 dec C rather than 1 hour at room temperature. It can strongly increase the signal (although I don't often have to do this).
3. Depends on the antibody- remember it's binding the protein, not the PVDF. Different Antibodies have different affinities for their antigens. A good antibody though will have an extremely strong affinity for its target and would probably survive a few days in TBS-T (although it's not ideal and you'll probably get a lower/messier signal). You should try to finish your blots over 2, maybe 3 days (if doing a prolonged secondary incubation).
We store our blots after we've finished by dehydrating and freezing them, although I've never actually had to go back and try to re-develop one again so I don't know how well that is actually tolerated. It's just something we do in case we needed to go back to a crucial experiment.
4. Same as 3, depends on the antibody.
Are you re-using your antibodies? You can get away with using an aliquot a couple times (maybe 3 with a primary, a few more with a good secondary or loading control primary) but you'll see a definite loss in signal intensity each time, and eventually you'll just stop getting anything.
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