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Position: Discussion forums > Bioscience biotechniques discussion forums > Immunology forum and cytometry forum
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Raising Antibody problems
Posted by: viju (IP Hidden, New member, 2)
Date: September 28, 2005 03:10AM
Hello everybody
I am facing problem in raising antibody against 35Kd recombinant protein in Rabbit. Immunization shedule is such that after primery immunization I give 1st booster after 21 days, and further boosters so on after every 21 days. I injected 80-100 microgram of my protein from SDS-PAGE gel band via subcutaneous route. I have checked my sera taken after 3rd booster by western, with His-Tag antibody as control as my protein is His-Tagged, but there is no specific band even in 1:250 dilution. what might be reason behind such response by Rabbit. Thanks. Vijaypal Yadav
Re: Raising Antibody problems
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: September 28, 2005 09:35AM
> I have checked my sera taken after 3rd booster by
> western, with His-Tag antibody as control as my > protein is His-Tagged, but there is no specific > band even in 1:250 dilution. What do you mean "with his-tag as control"? Are you using his-tag antibody and anti sera on a blot with your recombinant protein? Typically the peak of antibody titer comes at ~180days but may vary. A less than 1:250 titer is very low indeed after the 3rd boost. If your recombinant protein is pure enough you should try to inject the soluble protein. Sometimes the antigen is difficult to release from the polyacrylamide gel after subcutaneouse injection. To check protein purity stain the gel with coomassie blue and you should only see one band if you load ~15ug. If your protein is 90% pure you may want to try soluble protein inject first anyway and boost with gel slices. The non-specific cross-reaction will decrease with each boost.
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