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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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speckled background on westerns
Posted by: Heather (IP Hidden, Unregistered user, )
Date: February 25, 2005 11:48AM
I am getting an odd artifact ofrom a new set of antibodies I am running on protein samples I have done Westerns on with 5 other antibodies without this problem.
When I develop the blots the bands are there and very nice and the background is low except for random specks andtiny smears all over the membrane that are darker than the bands themselves. All reagents and primary antibodies are new. Samples are prepped by boiling @ 98 C for 5 minutes with loading buffer prior to loading. Membranes blocked overnight in 3% milk protein in TBST. Transfer appears great (checked with ponseau S prior to incubation with block and primary Ab). I have used the secondary antibody before with no blotching occurring (it is HP bound). Any ideas how to stop this? The company said it could be anything.... I am very irritated and I don't have limitless sample to just keep redoing them.
Re: speckled background on westerns
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: March 14, 2005 11:56AM
Are you using fresh blcoking buffer every time? do you have sodium azide in the blocking buffer to limit conatmantion? Are you using Nitrocellulose or PVDF membranes)
I think blocking overnight is exssive, you can try for 60 minutes and I am sure the tiny specs will diseapear. Many people think that these specs are primerly points of static charge accumelation especialy on NC membranes, if this the case reducing the shaking speed might help reducing this phenomena. Good Luck
Re: speckled background on westerns
Posted by: Heatherbee (IP Hidden, New member, 3)
Date: April 1, 2005 07:21AM
To answer your queries:
-I do use fresh block solution every time, but I don't have sodium azide in the blocking buffer, just in the blocking buffer I use to make up my primary antibody solution. -I use PVDF membrane (seems to give me a better transfer) -I block overnight as it is the most convenient thing - by the time I prep samples, quantify and run the gels and blot to the membrane it is the end of the day and I can block overnight and begin the antibody incubation first thing the next day. I'll try turning down the shaking speed. The company has sent new antibody for the same protein and it has none of the artifacts I saw previously. I suppose it was the antibody as the new antibody is working fine. Thanks for the tips. I'll keep them in mind if I have similar problems again. Edited 2 times. Last edit at 04/01/05 07:23AM by Heatherbee.
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