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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.


Western Blot suddenly stopped working!!! :(

Western Blot suddenly stopped working!!! :( - Western Blot Forum

Western Blot suddenly stopped working!!! :( - 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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Old 06-24-2011, 04:41 PM
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Unhappy Western Blot suddenly stopped working!!! :(



Hello Everyone,

I am a new user to this forum and need help. I am a phd student for 4 years and always have (despite the odd one) perfect westerns.
Sometimes is the diH20 cartridge needs replacing, the impure water can cause signal problems.
This happened recently so once the filter was replaced I presumed they would work again, as this has been the case before.
But I am still getting no signal! I have thrown away all buffers, ALL buffers and made them fresh and nothing!
I am at my wits end.. please help!

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Old 06-27-2011, 03:18 PM
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Smile Re: Western Blot suddenly stopped working!!! :(

Quote:
Originally Posted by biotoxgal View Post
Hello Everyone,

But I am still getting no signal! I have thrown away all buffers, ALL buffers and made them fresh and nothing!
I am at my wits end.. please help!

try a dot blot with a positive and negative control, to test your antibodies and color detection system. All you need to do is spot the controls on the membrane and incubate at 37C until complete dryness/can use pencil to mark the controls on membrane--then rely on your WB protocol for detection.

If this works check your running buffers for pH, electrophoresis apparatus, loading buffer (these can go bad due to pH, contamination, temp fluctuations).
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Old 12-31-2011, 11:47 PM
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Default Re: Western Blot suddenly stopped working!!! :(

Probably its your antibody going bad!
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