Quantification of steptavidin coating - (Feb/09/2007 )
I am coating the microwell plate with streptavidin using adsorption method. Can anyone suggest me a relaible protocol to quantitate the amont of streptavidin getting coated in the microwells?
-Loribio-
When you do an ELISA using biotin-HRP, you can quantitate the amount of molecules enzyme bound from the OD you get out of your reader. There are a certain amount of biotin-HRP molecules that bind to streptavidin, so based on the OD you can find the amount of coated antibody bound.
-WAstate-
QUOTE (WAstate @ Feb 9 2007, 09:00 AM)
When you do an ELISA using biotin-HRP, you can quantitate the amount of molecules enzyme bound from the OD you get out of your reader. There are a certain amount of biotin-HRP molecules that bind to streptavidin, so based on the OD you can find the amount of coated antibody bound.
Thankyou very much for your suggesstion. But then my problem is how do i prepare standered curve for it. Because as i feel even if am putting known quantities of streptavidin in the well for coating, i am not sure how much of it is actually present in well after whasing and all.
Kindly help me.
-Loribio-
You can google the ratio of biotin-HRP that binds per streptavidin molecule, or some data sheets state it. You know how much you put on the plate, then quantify how much enzyme from the ELISA reading and compare.
-WAstate-