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Annoying occasional western problem ... anyone have any explanation?


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#1 theseeker

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Posted 06 April 2010 - 05:51 PM

After lots of trial and error, I have my western blots working generally quite well. I use a pretty standard BioRad system with semi-dry transfers.

I always do them exactly the same way because, as I said, they usually work quite well. I use fresh buffers/reagents almost every time to avoid wasting time on blots that don't turn out right.

There's a very annoying problem that occasionally crops up that I wonder if anyone who's more of an expert on immunoblotting can explain. Every now and then, a set of membranes will become completely resistant to having a durable signal after the application of ECL reagents. They'll give a pretty normal signal for the first minute or two after the ECL is applied but then, it will completely fade thereafter if I need to do a longer exposure. Literally two or three minutes after the ECL is applied they completely stop giving any signal whatsoever. Once a membrane starts doing this, it will do it for the rest of its life, regardless different batches and brands of ECL. So it's definitely not the ECL itself. It's also definitely not something with the nitrocellulose membranes before use, as I always use the same stock of precut membranes that are kept in our lab. This also happened a couple times to another lab member, and she wasn't able to figure out the cause either.

Anyone have any guesses?

#2 RUFY3

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Posted 07 April 2010 - 05:56 AM

Hmm interesting .. I also faced the same problem few months before in my lab and wasnt able to figure out the cause... so i had run the WB again and it worked fine....but anyone there who has found the reason/solution for this problem can please answer this post.. :P

#3 mdfenko

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Posted 07 April 2010 - 07:19 AM

too much protein can cause this. this leads to too much secondary antibody conjugate binding and it uses up the ecl substrate too rapidly.

how is the background? maybe the blocking wasn't optimal so you get too much secondary binding over the entire surface.
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#4 theseeker

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Posted 08 April 2010 - 04:50 AM

too much protein can cause this. this leads to too much secondary antibody conjugate binding and it uses up the ecl substrate too rapidly.

how is the background? maybe the blocking wasn't optimal so you get too much secondary binding over the entire surface.


Background/blocking doesn't seem to be the issue. The amount of protein shouldn't be the cause either as I pretty much always load gels the same (about 40 ug per lane using a 10-lane comb).

I don't know, it just seems to come out of the blue, as though I've angered the gods of western blotting. :o

#5 fysio lab

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Posted 27 April 2010 - 04:48 AM

idem dito here, i've had the same problem, thought it was because we have barrels filled with aqua dest. Since we are a small lab, sometimes they are standing there for a month..i suspected growth and never used water from it again. problems were solved...coincidence?????
Good luck with finding the reason.




too much protein can cause this. this leads to too much secondary antibody conjugate binding and it uses up the ecl substrate too rapidly.

how is the background? maybe the blocking wasn't optimal so you get too much secondary binding over the entire surface.


Background/blocking doesn't seem to be the issue. The amount of protein shouldn't be the cause either as I pretty much always load gels the same (about 40 ug per lane using a 10-lane comb).

I don't know, it just seems to come out of the blue, as though I've angered the gods of western blotting. :lol:






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