Hello Everyone,
We have a secondary antibody that we biotinylated for detection in our ELISA system that is Strep-HRP and TMB based.
However the his-tag is on the C-terminus and we did not get ride of it because we did not think it was an issue. Now however, we think the His6 is causing non-specific binding of the Strep-HRP resulting in high background (i.e., even when there is no sample/antigen).
Currently our wash buffer is only 1XPBS. Can we keep it simple and add a detergent (e.g., 0.05% Tween-20) to the buffer to reducing this issue. Removing the tag is 20 steps backward and alters protein performance.
Thanks all
If the his-tag is causing the problem, you should be able to use imidazole to block it. 40 mM imidazole would quench non-specific bindings, and 250 mM would quench a specific interaction, such as HRP's metal, with the his-tag. If that has no effect, it would suggest that the problem is likely somewhere else.
I'm guessing you have already tried varying the antibody concentration...?
Also, why do you have a his-tag on an antibody in the first place? Those should be purified on protein A, not with his-tag...
Each situation is somewhat unique so it's hard to predict what will solve the problem. Probably easiest to just set up a bunch of controls and test various conditions. Adding Tween is a reasonable thing to try. You might also try reducing the amount of your 2nd Ab you use, it might be too high. It could be your blocking step is not adequate so double check that and also make sure that you are doing adequate washes after your 1st Ab.
If the his-tag is causing the problem, you should be able to use imidazole to block it. 40 mM imidazole would quench non-specific bindings, and 250 mM would quench a specific interaction, such as HRP's metal, with the his-tag. If that has no effect, it would suggest that the problem is likely somewhere else.
I'm guessing you have already tried varying the antibody concentration...?
Also, why do you have a his-tag on an antibody in the first place? Those should be purified on protein A, not with his-tag...
John Schloendorn
Hi John,
I meant nanobody not antibody. I alway slip up since I only use Antibodies (generally).
Hi John,
One more follow up. The capture has a His6 as well. We find that it is better able to stick to the Nunc plate compared with no tag or strep coating plates. Your suggestion of Imidazole sounds great, but I worry that such a concentration would release the capture from the 96-well plate.
Best,
Norelle
Are you certain the the problem cuased by the secondary his tag?
You have already his tag on the caprture with no problems.
Many times the biotin strepavidin creating complex that cuasing irrelevant signal. Add detergent to 0.05% to the wash. I would suggest direct labeling of antibody with HRP.
Good luck
Oded
John Schloendorn
So it looks like the his tag of the detection nanobody is causing it to bind non-specifically to the plate (even though it has already been blocked with 2% BSA).
This then causes STREP-HRP binding (detection Nb is biotinylated).
To eliminate this effect, would the Imi idea you proposed still work?
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