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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Light areas or no signal at all.

Light areas or no signal at all. - Western Blot Forum

Light areas or no signal at all. - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 06-30-2011, 09:31 AM
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Default Light areas or no signal at all.



Hi, I am new to this forum.

In our laboratory we experience some weird problems regarding the Western blot protocoll we used for years.

It all started about 3 month ago. We didn´t get any signal with the normal ECL (GE) we use.
With the enhanced ECL (Millipore) we mostly get excessive signals but not as long as normal.
When we put a film on, 5 min after appling the ECL there is no signal.
In some occasions we see „light“ areas (like clouds).
And the keep staying at the same place everytime we try another ECL (so first standard ECL, then wash with TBS-T, then enhanced ECL -> Same clouds)

We got a fresh Nitrocellulose (Millipore), ECL (another Lot), non-fat milk (changed from noname to Bio-Rad, which seemed to work better… but not for long).

I tryed reducing the milk concentration from 5% to 3% with the old milk – that appeared to be a little bit better. But with the new Bio-Rad milk we continued to use 5% since it normaly should work. It appeared to work with the new milk.

But the problems continue… Anyone any tips?

Thanks,
Daniel
Munich
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Old 06-30-2011, 07:47 PM
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Default Re: Light areas or no signal at all.

Can you post a picture of the films? It might help to diagnose the problem.

I bet the problem is not in the ECL reagents (especially if you have used two different products).

How old is your secondary antibody? I have been told that the HRP activity can decrease over time (although we use secondaries that are at least several years old with no problems).

Do you stain the nitrocellulose after transfer (Ponceau S) to make sure the blot looks OK??

I didn't quite get the last part: you say the Western works with Bio-rad milk but not another brands??? Dry milk can be quite variable between different sources and some antibodies may be sensitive to specific components. I know some dry milk is not totally non-fat and this could affect performance. You might also try BSA block if this seems to be a critical point.
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Old 07-01-2011, 11:41 AM
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Default Re: Light areas or no signal at all.

tinyurl.com/65pzseh (I am not yeat allowed to post URLs...)

The HRP-antibody is about 3 month "old".

Ponceau S and Coomassie blue stainings both look good.

Well, I meant the WB "works" with enhanced ECL with 5%milk-TBST (Bio-Rad) - well at least I get some kind of signal.
I don´t think the milk is the problem. The "better" results are not that reproducible.

The antibodys are replaced a such a high rate, so it´s not because they are old.

One more symptom now is, that the normal ECL´s signal disapears too fast. After 1 min the signal is gone.

Thank you, so far.
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Old 08-24-2011, 07:18 PM
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Default Re: Light areas or no signal at all.

Have you tried to detect "easier" protein, e.g. GAPDH, beta-actin...? If they came out fine, it's probably your dilution...
Do you have background? (cloudiness but not bands)...
Or try to increase the protein loading concentration, maybe...

Well, good luck!
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Old 09-24-2011, 07:46 PM
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Default Re: Light areas or no signal at all.

Hi,
We have been having similar problems so I was wondering if you ever figured out your problem because we have had the same thing where we have used the same ECL procedure for years with out problems.
We always stain with ponceau and the proteins look evenly loaded and you can tell there is a lot there. But the problem tends to vary, some times just certain parts of the film get light area and some times the ECL can be seen on the blot with really strong antibodies, but then it doesn't show up on the film. However it's not consistent. We've done bolts in parallel, run on the same machine, blocked with the same milk, stained with the same antibody dilution, stained with the same secondary, and used the same ECL. Then Exposed the blots on the same film and one has worked and the other hasn't, but by all accounts they should have been identical.
We have tried new ECL, new secondaries, and nitrocellulose (all from other other labs who aren't having problems) and new antibody dilutions. We have switched out all the buffers and gotten different water to make them from different Milli Q water (also from people who weren't having trouble). We have also tried different developers and that hasn't solved the problem either.

I know it's not the exact same problem as you, but I was hoping you had found something else out that could help.

Thanks!
Kari
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