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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Goat Polyclonal Antibody in Westerns
Posted by: F.A (IP Hidden, New member, 2)
Date: January 22, 2005 06:14PM
Hi,
I am having trouble with high membrane backgrounds when using primary goat polyclonal antibody. My membrane is dark and the bands in my test samples are very faint while my positve control is ok, probably because it is transfected with the protein in question. I incubate my membrane with 200ug/ml of goat polyclonal antibody in 1% milk with Tbst overnight. The following morning I wash the membrane 3 times with 1% milk for a total of 30 minutes. The secondary antibody is donkey anti-goat IgG HRP which is added at 1:2000 and incubated at room temperature for 2 hrs followed by three 30 minute washes with 1% milk with TBSt and a final fourth wash with PBS for ten minutes. Any suggestions on how I can get a cleaner membrane with strong band signals? My TBST contains 0.01% tween 20. Thanks....
Re: Goat Polyclonal Antibody in Westerns
Posted by: CD197 (IP Hidden, Unregistered user, )
Date: January 23, 2005 01:06PM
"I incubate my membrane with 200ug/ml of goat polyclonal antibody in 1% milk with Tbst overnight." ????????
The reasonable final conc. of Ab should be 0.1-1 ug/mL (usually one vial of antibody total only has 100-200 ug)??? Dilute further your goat Ab.
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