AP vs. HRP secondary

[sarahstudeman]

Hello all!

I have a primary antibody that I'm getting really low signal from. I have done several things to increase the signal strength, including increasing antibody concentration, increasing incubation time, and decreasing the concentration of blocker in the antibody solution. These all resulted in a mild increase in signal and a significant increase in non-specific background. We are currently using an HRP secondary and detecting with supersignal. I've been doing overnight film exposures and getting clear but VERY faint bands. The protein concentrations should be adequate, and increasing the concentration of protein in the samples would be very difficult.

I remember long ago when we were first starting to run Western Blots, we were getting an excessive amount of signal strength probing for a different protein with an AP secondary using the SuperStar system. We got less signal when we probed the same primary Ab with an HRP secondary. We aren't sure if that was a product of the AP system or if it was something funny we did due to our lack of experience. (Btw, I'm not kidding about signal strength. You could see the bands with the naked eye. It blackened our blots instantly.) I was just wondering if this was something anyone else had experienced? I'd like to get some conformation on my theory before I go shell out the money for a new secondary and detection system.

Thank you all!

[Tfal]

well I'm just a WB novice and I have never tried an AP, so I have no answerfor your poll.
But I have been having similar problems with my blots (with HRP). Here are things I was told to try or I found around and tried out.
Have you tried much longer blocking, say overnight? (If you do ON, at 4 C).
Have you tried a different blocking agent? Is your blocking agent different between your Abs incubations and blocking per se?
How about increasing wash time or number?

I hope you tell us how it turns out if you change to AP.

Anyway, Good luck.
Tfal

[sarahstudeman]

I haven't tried blocking overnight. I'm just afraid that I'd lose what little signal I have. Also, adding another overnight step would mean it would take me three days to run a WB, which would bum me out. My problem is that everything I do to get rid of the background decreases my signal...

I'm going to try to increase the NaCl concentration in the PBS next time I use that Ab. I read that somewhere. I made it 0.3M, which is almost triple what we normally use. However, I haven't had an opportunity to use that primary since I posted this because we went back to working with a different protein for awhile. I'll let you know if it works for me.

[Bonnie]

I recently experienced an antibody where we got almost no signal when we blocked with 5% milk, but when we blocked with 3% BSA instead, the signal was bright and clear.

So definitely consider whether your blocking agent may be masking the antigen.