Fri Jun 23, 2017 6:27 pm by GregBehbehani
Hi Marjolijn,
I suspect that your tetramers are binding to the beads non-specifically. These type of beads will readily bind any protein, so if there is any free protein in your sample it will stick. This may be because the tetramers have a higher off rate and so come off of your sample to a greater extent than the other antibodies that you're analyzing. Obviously, the anti-APC and anti-PE antibodies themselves are also proteins and could be binding directly to the beads or binding to the beads while bound to your tetramers. I don't have a great explanation as to why PE binds better than APC, but I doubt it matters that much.
I'm also not as sure why your lineage markers are getting stuck to the beads, I don't think I ever seen this. I would try to be sure that you're washing your cells thoroughly after antibody binding and that your post fixation (typically during intercalation) is adequate. If you're only seeing the lineage markers on the beads (i.e. no Ir signal) then I would think this could only be coming from free antibody that has fallen off of your cells. You could try saving the supernate from each of your post-staining washes, then diluting and running it in solution mode (you'll have to empirically sort out the right dilution, but it will probably be a low nanomolar concentration of staining antibody). This should allow you to directly see how much antibody is falling off of your cells at each step of your protocol.
Fortunately, the solution to your problem should be easy. I would suggest that you try pre-washing your EQ beads in a protein containing solution (e.g. cell staining media) then wash a few times in water, then add them to your cells. I would bet that this will greatly reduce the non-specific binding that you're seeing. But it would be nice to do the experiments above, as you'd probably like to sort out why so much of your antibody is falling off your cells.
Let us know what you find out.
best,
Greg