Mon Sep 26, 2016 3:29 pm by Chowduck
Hi S.J.,
I would back off the 300ug a little, your staining might be "dimmer". I use 150ug as a balance of risk/reward that I’m comfortable with. Here are some calculations you may find helpful:
Polymer: 11kDa, 100ug/rxn, (~10 chelation sites/polymer) = 9.09x10^-9 mol polymer/rxn
Antibody: 150kDa, 100ug/rxn, (4-6 conjugated polymers/antibody) = 6.67x10^-10 mol Antibody/rxn
Metal: 50mM, 5uL per rxn = 2.50x10^-7 mol metal/rxn
Per reaction this means that there are 13.64 available polymers per antibody, at the high end if we expect 6 polymers to bind then this would saturate at 227ug of input antibody assuming the reaction exhausts the entire supply of polymer. (If 4 polymers then 340ug of antibody, but why aim low. Also you might be left with free -SH groups). At 5uL the metal is in a 2.8 fold excess to chelating sites on the polymer so don’t worry about that.
Another observation I’ve made is that most of the antibody loss is going to happen in the first few spins meaning that if you’re loading 300ug, by the time that antibody comes into contact with the polymer you might be looking at ~250ug remaining. If the recovery is poor and I have starting material remaining I will run ~5-10ug on an SDS-PAGE to get a sense of purity or presence of peptide fragments that may wash through the filter. I’m not certain why the increased volume affected your recovery unless there was a filter specific defect. If you repeat it, pull some antibody out after the reduction, prior to the conjugate to look at on a SDS-PAGE maybe it was over-reduced?
Regards,
-Greg
UHN/Sickkids FCF
Toronto, Canada