Hi,

I woud recommend using Biacore system (if you have in your institute or if you know a lab that have it). The material is not very expensive. In attach, i send you a brief introduction to the system.

If you need any help please feel free to contact me.

Good work.

Hi,

one of the classical assays for your purpose is a microtiterplate binding assay. In brief, one binding partner is recombinantly expressed and covalently bound to the surface of a microtiter plate. The second binding partner is in vitro transcribed+translated and radioactively labeled. The labeled protein is incubated with the immobilized protein, then the ratio between bound and unbound protein is determined at various concentrations.

The recombinant expression of protein in bacteria is relatively cheap, the in vitro translation and labeling may cost something like 500$.

Best wishes,

Andreas

Hi Andreas,

I think radioactively labeled method is very dangerous and my laboratory cannot supply enough safety equipment for this protocol. So, can I use monoclonal antibody of second protein as an alternative material ?

The second thing that is I want to measure Ka and Kd in comparison with reference.

So, Ka and Kd is neccessary to identify.

Hi Thang,

the problem is, if you detect with an antibody it's not quantitative anymore. The big advantage of radioactive labelling is its sensitivity and it allows quantitation via a scintillation counter (-> for determining the Kd value).

The amount of 35S-methionin that is used in such assays is very little, and 35-S is also not so critical as other isotopes because it is easy to shield (its mainly beta irradiation) - but of course you have to have a special training to handle radioactive material.

You may google if there's an alternative labelling technique available (via luminescence probably?) which allows a quantitative readout.

Best wishes,

Andreas