Hello All,
Is it possible to re-developing signal without stripping in Western blot? and How do you do it? Please share. It has been stuck in my head for a while. My objective is just to re-develop signal the same protein on the membrane again, in case ECL expired or want to capture image again. No need to change primary and secondary antibody.
From what I read and heard, there are three ways to re-developing signal in western blot:
1. Stripping, blocking, and then re-probing primary and secondary antibodies again, then re-developing with ECL -> lost some protein sample. This method is written in every protocol.
2. Don't strip, just re-probing with secondary antibody, then re-developing with ECL.
3. Washing membrane in TBST for a few time, then re-developing with ECL. -> heard from lab mate -> haven't found reference in google.
Have you ever tried 2nd and 3rd method? Does it work? Please verify
Thank you very much,
If you had seen the signal previously by probing with your secondary antibody means that epitopes on the primary antibody is occupied by secondary antibody. If you probe it again with the same secondary antibody which is already bound on your membrane may result in no binding and you may see the same result again as previously or it can also increase the signal intensity which is also possible if you probe again with your same secondary antibody.
If you want to redevelop the signal and you had seen the signal previously, you can just scan the same membrane again after re-developing with ECL will give you the same results again because the secondary antibody conjugated with HRP is already bound and you are just providing the substrate to the enzyme HRP which is again in the same form after the luminol compound in your ECL kit converted to APA and produced the light . If you are developing the X-Ray film, then re-developing in ECL reagents will increase the background.
If you did not see any signal means that either the ECL reagents did not work or dilution of secondary antibody is not ideal or the antibody used against primary antibody is not specific to the host in which the primary antibody was raised. In first case where ECL reagents did not work, just look at how much old your reagents are and re-develop again. In the second case of dilution, you may re-probe again with either more concentrated secondary antibody or with the same dilution again and you may see the signal. In third case, the stripping is required.
The third point of washing the membrane with TBST and re-developing again with same ECL reagents will yield the same results again with less background. And depending on your previous results, if you want to see the same signal again as previously, then you can go for it.
I think stripping is primarily for when you need to re-probe with a different primary antibody. I've never had very good luck with the strip blot. #2 and #3 are easy to test though, just try it next time you're probing a blot and let me know if it works =) If you want to probe for a different antigen/epitope on the same blot, I suppose you could just use another primary antibody from a different species (e.g. rabbit) and a secondary with a different enzyme (e.g. AP). I haven't tried this either...
If you had seen the signal previously by probing with your secondary antibody means that epitopes on the primary antibody is occupied by secondary antibody. If you probe it again with the same secondary antibody which is already bound on your membrane may result in no binding and you may see the same result again as previously or it can also increase the signal intensity which is also possible if you probe again with your same secondary antibody.
If you want to redevelop the signal and you had seen the signal previously, you can just scan the same membrane again after re-developing with ECL will give you the same results again because the secondary antibody conjugated with HRP is already bound and you are just providing the substrate to the enzyme HRP which is again in the same form after the luminol compound in your ECL kit converted to APA and produced the light . If you are developing the X-Ray film, then re-developing in ECL reagents will increase the background.
If you did not see any signal means that either the ECL reagents did not work or dilution of secondary antibody is not ideal or the antibody used against primary antibody is not specific to the host in which the primary antibody was raised. In first case where ECL reagents did not work, just look at how much old your reagents are and re-develop again. In the second case of dilution, you may re-probe again with either more concentrated secondary antibody or with the same dilution again and you may see the signal. In third case, the stripping is required.
The third point of washing the membrane with TBST and re-developing again with same ECL reagents will yield the same results again with less background. And depending on your previous results, if you want to see the same signal again as previously, then you can go for it.
Both the 2nd and 3rd methods will work.
I usually simply add more ECL reagent to the blot and do another exposure if the blot is already clean, or just rinse with plain TBS and repeat the ECL reaction. The presence of Tween20 (detergent) can promote bubbling, which you are trying to avoid.
2.Don't strip, just re-probing with secondary antibody, then re-developing with ECL.
I choice this method when my blot is old (several weeks).
3. Washing membrane in TBST for a few time, then re-developing with ECL. -> this is possible when the blot was done in the week because HRP is active.
If you want to re-capture the signal, you do not need to add primary and secondary antibodies again, just add new ECL.
If the membrane is old and has been kept at room temperature the antibodies or the HRP can be degraded and then the ECL will not give you a signal. In that case you strip
Jose, I've found that even when stored in the fridge it's hard to preserve the HRP conjugate after more than a day or two, but you make a good point. I will only strip before probing with a different primary antibody though; I find that it's sufficient to repeat the secondary on an old blot.
no need to strip if its the same antibody. Either redoing primary and secondary or just secondary would work.....if there is an intense signal then just redoing ECL will work, but this may depend on how long after the original blot was probed...if it was the same day then it will be ok....any longer then the detection will be reduced if just adding ECL to the same blot.
The HRP conjugate will lose activity over time, so just adding new ECL substrate may not work well. You can re-incubate with HRP secondary to re-detect the blot - but some of the epitopes on the bound primary will be occupied, so quantitative analysis is probably not a good idea.
If your signal is ok, you do not need to incubate in primary and sec Ab. Just wash with TBST for a short time and add new ECL reagent.
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