Simply titrate antibodies concentration against an antigen by ELISA (coat 100-200 ng of your antigen pure/impure), block it and use 1;100-1;25000 dilution of yr antibodies. Ofcourse it depends on the concentration of your antibodies and purity. They work better if they are affinity purified. If you wanted to see the specificity under denaturing conditions then you can do western blot.

Thank you very much for your help

and what do you think about immunoprecipitation?

I think immunoprecipitation can also performed, you can see the complex in gels, but its more sophisticated, less ssnsitive and less precise than ELISA. of course you should have secondary antibodies-HRP conjugated for characterising primary antibodies. You can use 2.5% BSA +0.05% Tween 20 in PBS as binding and blocking buffer. Use 0.05% Tween 20 in PBS for washing. its easy ans streight.

Your help is very important for me,thank you

And do you use antibodies of integrins blocking?

Hello again,

We are working with almost all integrins that are interacting with ECM proteins, like vitronectin, fibronectin etc.... Some times antibodies for integrins can block the interacting ligand/protein. But it cant be the same with all. integrins are big surface structures and oftem demerises when recognise the ligands. So the binding regions for various ligands may vary, if the ligand binding regions are masked by your antibodies, then it will work in blocking. Another problem is anti-integrins are very expensive, so you can use them only for a limit.

If you want to block integrins binding to some proteins....like if RGD interaction motif is involved, you can use commercially used RGD peptides, that works perfectly.

The quickest, easiest and cheapest way is to radio label the Ag with 125 Iodine (gamma emitting isotope) and use Radioimmunoassay curves to characterise binding. You can measure titre, affinity, binding site number (Scatchard) , specificity, cross reactivity and other parameters. The RIA will enable you to detect Abs and measure the native antigen. You can use the radioligand to locate and characterise binding sites by auto radiography. Send a few micrograms and I can label it for $600 and send it back the next day. [email protected]

Radio-immuno assay seems to be easier one but need careful handing and trained personals to carry such test due to nature of radio study