Help with Streptavin coated plate - (Dec/02/2008 )

Dear all,

I am a student starting to do ELISA. I just cannot get rid of the background signal. Need some expert advice.
I coat 96 well plate overnight with 12ug of streptavidin in PBS (I want to saturate the well)
Then, I washed 3 times with TBST
Block with 5% Milk in PBS for 1 hour
Add 20ug of Biotin-HRP in 5% milk for 1 hour
Wash 3 times
100ul of TMB
100ul of H2SO4 to stop the reaction
Read absorbance at 450nm

When I re-do this protocol on empty wells (no strep), I have super high signal (comparable to streptavidin coated wells).
Are there any problems with the protocol? I had also tried BSA
Help.. Thanks guys!

Is that 20 ug HRP in each well? That sounds very high. Have you tried simply diluting it? And diluting your strep, while we're on the subject?

Milk contains biotin, so you might be saturating your streptavidin during blocking.

Hi, Thanks for the advices!
My purpose is to find out which level of biotin-HRP will give me a maximum signal (assuming I had saturate the well already with 12ug of strep). Therefore, I used such a high amount of biotin-HRP.

ALthough using all the steps trying to saturate the well, I still get very high background with control wells (ie, wells without any streptavidin coating). I didnt know that milk contains biotin... Thanks!
Any idea what level of strep is comfortable to saturate a 96 well plate? and which blocker is best to reduce signal so no non-specific bitoitn-HRP binding?

For blocking you might try BSA (purity can be important), gelatin or commercial solutions.
For example: 1-5% BSA/0.05% Tween-20/PBS
Washing steps can be increased as well.

As for coating - it depends on the plates, so check the manufacturer's instructions.
Although SatanClaus is right - less is better.