I run a western blot for iNOS using GAPDH as my housekeeping protein. I used ECL, following manufacturer's protocol, exposed it with X-ray film for 5 min, and got a very pretty band for the iNOS, but not quite good with the GAPDH. I re-exposed the membrane longer for 10 min, but the bands were fainter. So, I decided on adding fresh ECL, then re-exposed the membrane for 10 min. Still, the bands were fainter. I incubated the membrane in TBST at 4'C overnight. The following day, I place fresh ECL exposed it for 10 mins, but the nothing was detected. As in NOTHING. Not even a slight background. The film was crystal clear. Tried it a couple of times, but no success. I decided to re-probe the membrane - w/o stripping w/ the 2ndary antibody. Washed it then exposed it for 10 min. Still NOTHING. What I did next is to repeat the blocking w/ 5% milk, incubate it w/ the primary and secondary antibody again, w/o stripping the membrane. Still no success in seeing any signal from the same membrane.
Does anyone have a thought on what's going on here? I had produced a very decent band for the protein on the first run, but to no avail in the succeeding exposures. Will appreciate any suggestions or thoughts regarding this.
In our lab we use ECL from two different providers. One has similar effects to what you describe and the other does not. Try repeating the experiment from scratch and comparing two different ECLs.
I have had successful blots after stripping and reprobing the membrane for GAPDH. Can I ask why you don't want to strip the membrane?
The ECL I use has a very short duration and what happen with my ECL is very similar to what happen to you: when I have a strong signal the duration is longer but when the signal is weak after the first exposition, I can't see anything after the second exposition! But I don't thing the problem is the link primary-secondary antibody. I suggest you to strip your membrane and incubate again with fresh antibidy.
Good luck!
@Gopika, stripping the membrane is not an issue. I was just wondering what happened to the membrane, which seems to have been stripped when all I did at first was to add ECL and re-expose it.
@Valeria, thanks. I'm in the process of doing that. I will update evryone if it worked or not.
Iused for iNOS, eNOS and nNOS western blots by colorimetric and ECL methods in our laboratory. But I used ECL plus because it have higher signal power than ECL. it is important signal duration. I think that you add ECL solution after you wait at least 10 min. what is the protein concentration of samples? you should incubate membrane with primary ant,body at overnight.
Of course iNOS it's difficoult to detect... But you also have problem with the housekeeping! Is it right? In any case there are different type of ECL: ECL with a longer duration, ECL used to detect small amount of protein with short duration! The Ideal would be to have the right ECL for every situation! Anyway using fresh primary antibody overnight and one longer exposition ( don't do a first short exposition but only one long exposition) I think you'll see your protein! (I hope!). let us know how it goes!
@Recep, i loaded 22 ug of protein per lane. I freshly mixed the ECL right before I add it to the membrane. I tried incubating the ECL for 1 and 3 min before wrapping it and exposing it. I incubated the primary overnight as well
@Valeria, Thank you for your inputs. I will consider looking into the various types if ECL.
@Mark, thanks again!
Hi Ryan, I think you pretty much have all the advice you need but I should say that we also use "cheap and cheerful" ECL reagents (as opposed to buying more expensive products), and these are only good for one exposure. After that, it's always necessary to strip and re-probe the membrane. Hope you get things working well.
Just an update. I mentioned that i was in the process of stripping the membrane and reprobing it. After doing so, i still got a very faint signal, compared to my previous run, even when I exposed the film for 1 h. I would probably end up running a new gel and do the whole thing allover. Thanks for all your valuable inputs!
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