I realize you can buy streptavidin coated plates, but money is a little tight in the lab right now so I was hoping to find a more cost efficient method since we go through these plates very fast.
Hi Brian, make a 10ug/ml solution of streptavidin in H2O, put 100ul into the wells and leave then to dry ON. Wash and block with 0.1% BSA for 1 hr RT and you are ready to go.
I think it should be fairly straightforward although the specifications of your assay might require specific modification of some steps such as blocking a bit. As usual, it's best to test the commercial and in-house side by side with the same material to see whether you are getting the same results.
To begin, I would go for Nunc Maxisorb plates. Then, acquire purified streptavidin(you can ask for free samples from different suppliers to see what works and what does not), and test the saturation of the coating. I would start with 0.05 M Carbonate-Bicarbonate buffer (pH 9.6) and making a wide reciprocal dilution series of the SA to find the optimal or saturating coating concentration for your assay. You can also try coating on PBS as well, but higher pH usually works better for proteins.
For coating time and volume, I usually go for o/n at RT for antibodies/sugars/lipids at 100ul/well, or +4C for longer periods. Then wash with PBS/0.05% Tween20, block at RT for few hours with 150-200ul/well with PBS/0.5%BSA and you should be good to go with your assay.
Good luck!
Hi Brian, make a 10ug/ml solution of streptavidin in H2O, put 100ul into the wells and leave then to dry ON. Wash and block with 0.1% BSA for 1 hr RT and you are ready to go.
Thanks Dr. Stefansson. I didn't realize it was that straightforward
can u apply the above method of immobilization of streptavidin to glass beads?
And do u know the rough estimation of the amount of streptavidin bound or the number of free biotin binding sites available per well
Cheers,
Yans
Juha Kotimaa and Steingrimur Stefansson
Do you guys not have a problem with high background due to residual strep on the plate that is not bound to an antibody or nanobody?
The reason is ask is because our detection nanobody is biotinylated. So the running hypothesis is that some nanobody is not binding the antigen, but rather the plate because of the strep coating.
Hi Norelle C. Wildburger Doing a control in the presence of 1 uM biotin should take care of that.
Hello,
I am looking for a Streptavidin-binding protocol as well, but I was wondering if anyone knows whether the type of plastic matters? For example, I am looking to coat polypropylene PCR tubes with Streptavidin, but most 96-well plates are polystyrene. Any help with this would be greatly appreciated!
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