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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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high background and white bands in western blot
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: August 23, 2005 07:02AM
What could be the cause of very high background with white bands in the western blot? All of a suddon I had this very strange problem. It is not because of protein overloading since it give good signal before when I loaded the same amount of protiens.
The basic steps: 15-30ug proteins were transferred on PVDF, air dried overnight, methanol treatment 15sec and immerse in water for 2min, 5% dry milk in TBS-T blocking for 1 hour, primary and secondary antibody (stable-HRP-conjugated from Pierece dura-pico kit) incubation and washing, detection with either Dura kit or pico kit from Pierece. Thanks for any suggestions.
Re: high background and white bands in western blot
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 24, 2005 12:03PM
White bands can indicate protein overloading as you know. However if you have a black blot (high background) with white bands, this is probably caused by too high antibody concentrations. Reduce the antibody concentration, especially the HRP conjugate. Signal that decreases quickly and the appearance of white bands are indications that there is too much HRP in the system.
Re: high background and white bands in western blot
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: August 25, 2005 05:57AM
The secondary antibody concentration is the same 1:10,000 as my control blot, which gave good and clean signal without any background. I will try to dilute the primary antibody from 1:1000 to 1:5000 as you suggested and will see what happens.
I am using rabbit sera that had been boosted three times. The second bleed sera gave good and clean signal at the same blotting condition. I added 0.02% azid to prevent microbial growing. I suspect something in the sera caused the extreamly high background. Anyone had similar problem before? Thanks.
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