These antibodies are working well for me separately but using then together is not providing me with a combined effect.
My first thought would to determine whether the two antibodies cross-react?
Do you know whether the antibodies have been cross-adsorbed. If they have not been cross-adsorbed you could pass them through a matrix containing immobilized serum proteins from potentially cross-reactive species
@Elisa:Is there a way to know if the antibodies have been cross adsorbed?We do not have a matrix to hold serum protein to pass the antibodies through.
@Ming Li-I think i should give this a try.You maybe are right.I have used slightly lesser pcAb than mcAb.
I used lesser because there is much lesser growth factor needed to be blocked by the polyclonal antibody.I have used enough recommended from Abcam to block 8 ng/ml of the growth factor while i only have about 50-100 pg/ml of this factor that i need to block.For the other growth factor where i used 10 ug/ml of the monoclonal antibody as recommended by R&D systems enough to block 8-10 ng/ml of the growth factor.
The supplier should give you that information. To improve the specificity of the Goat anti-human and remove potential risk of cross reaction with a mouse antibody you could cross link agarose beads to Mouse IgG (whole molecule) and pass the Goat antibody through.
Is the mouse monoclonal IgG1? Was the Goat polyclonal purified with protein A? on occassion when antibodies are purified with protein A using old columns, the column becomes leaky and protein A may end up in the purified antibody. In immunoassay when you mix a mouse IgG1 with an antibody contaminated with protein A it will create non specific binding of the IgG1 to the protein A, preventing binding to the actual target. The only way of getting rid of this protein A contamination is thorugh ion affinity chromatograpy.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies