Hi /sci/ Do you guys know if vortexting or sonicating a solution

>>8817874
Could the antibodies have come off the beads during sonication?

>>8817885
maybe, that's what I was wondering. can that happen? i thought sonication works on the micron scale and would not impact the nanoscale molecules

>>8817928
I honestly have no idea but I don't think it would be an issue. I'd check other variables first, e.g. incubation time. What did you use/how did you check whether there was binding?

>>8817939
Yeah before resorting to something we have no information on if it can even happen testing things we know can go wrong would be better. What temperature was the solution at by the way OP?

>>8817939
My secondary antibody has an enzyme called HRP attached to it. After vigorous washing, if there is a complex with primary-antigen-secondary-enzyme, the solution should light up.

If there is no antigen, there will only be primary antibody on the beads so no enzyme should be present. There will be minimum background fluorescence but the separation between the positive and negative should be substantial.

File: ELISA%20[1].jpg (19KB, 750x406px) Image search: [Google]

19KB, 750x406px

>>8817955
The solution was incubated at room temperature.

I'm basically doing as shown in the image. If there is no antigen, there should be no fluorescence.

>>8817874
I don't have 100% relevant information but when doing ChIP (binding a antibody to a target dna/chromatin) I have never had issues with vortexing.

>>8817969
Perhaps the ab-ag affinity is weak. Run incubation at 37 degrees? If nothing apparent binding occurs then you could rule that out as a cause

>>8817999
So incubation at 37C generally improves binding between antibody-antigen?

I think the Kd of my primary is the 10s of pM range and for the secondary it is in the 1nM range ish. From the paper I followed, they used room temperature incubation at 1h, so I followed that. But 37C should provide more diffusion and energy for binding to occur you think?

>>8818002
Room temperature and 37 are common temperatures in immunological assays such as the ELISA with HRP, any more and you probably risk harming the proteins. I don't know if it generally improves binding, but I can imagine for weaker less specific binding it may encourage a detectable signal where there was none before. To rule that out i think you have to do a mock physiological-temperature run

>>8817969
That's odd, these kind of tests are typically pretty reliable. Do you know if there's any possibility that the fluorescent antibody inhibits binding with the one on the bead?
>>8817999
This would be a better test to run first.