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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.


Control protein is nice, but target protein is blur and not sharp

Control protein is nice, but target protein is blur and not sharp - Western Blot Forum

Control protein is nice, but target protein is blur and not sharp - 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 09-01-2010, 05:36 AM
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Default Control protein is nice, but target protein is blur and not sharp



Hi, I am running my western blot for a protein around 20kDa, the control protein that I used look nice (b-actin 43kDa) but my target protein is blur and not sharp. Could it be transfer problem or sample degradation? if sample is degraded, will the control protein still look nice?
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Old 09-01-2010, 03:41 PM
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Default Re: Control protein is nice, but target protein is blur and not sharp

Most likely there is some degradation going on. Each protein has its own properties. Some are very stable while other are highly unstable.

Take as many precautions as you like to maintain your protein, like : avoid freeze-thawing cycles, use proteinase inhibitors, EDTA, buffer up to pH 8 (pH drops with temperature in most buffers), DTT.

I usually get nice bands on the Western despite what the gel band looks like; best strategy is not to load too much protein on gel, and have optimized Antibody concentrations/dilutions for the WB.
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