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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.


Signal loss from blot

Signal loss from blot - Western Blot Forum

Signal loss from blot - 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 09-10-2011, 02:06 PM
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Default Signal loss from blot



I have previously routinely stored nitrocellulose blots for up to 3 months at 4 degrees in TBST with azide with no loss of signal (we were using HRP secondaries and ECL development). I have recently started using the Odyssey system (fluorescence conjugated secondaries) and using their recommended buffers etc so am using PBS with tween 0.1% for washes and storage, again at 4 degrees. For some reason I seem to be losing signal very rapidly over time eg about a week. My initial phospho-blots are fine but after storage and re-blotting I can't detect anything. Basically even tubulin is virtually undetectable. If anyone has any suggestions or explanations as to what may be going on I would really appreciate it, its really driving me crazy!
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Old 02-15-2012, 04:37 AM
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Default Re: Signal loss from blot

Hi there,
some manufacturers may have interfering salts, buffers or even residual proteinases / chemicals in their reagents that may interfere or degrade the quality of proteins and their detection.

We have noticed the same thing with different buffers, antibodies, and blots as you have.

Of note you mention Phosho-blots and unfortunately that may be the key issue to your failure to detect the proteins with blotting at a later stage.

Phosphorylation is notoriously difficult to detect even with fresh antibody, protein and blots. The issue is phosphorylation is a modification which is easily modified by many phosphrylation enzymes, for example removed by phosphatase, and lost.

Were you always able to reblot phospho-protein blots (ie blotting for signalling detection) after storage or was it just protein detection?
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Old 08-20-2012, 10:38 AM
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