I've been working on similar, my antibodies came in 50% glycerol (from Cell Signaling) and have conjugated OK (hitting 15-25k MFI when I tested bead/antibody binding).  I had first tried antibodies from Abcam because they came without preservatives. They also conjugated fine (I had to scrap the Abcam antibodies later on in the optimisation process however) so I'd probably say that glycerol didn't have that great an effect.  How are you validating your bead conjugation?

Hi Adam,

Thanks so much for your response! 

That's interesting that you say that. Everything looked ok for ours as well until we tried to validate the beads by doing an inhibition assay. We are getting around 50-60% inhibition when the serum is incubated with protein. Also, tried saturating the serum with the protein, but to no avail. That was why we thought the glycerol may have been an issue.

Tabitha,

I usually dialyze my antibodies against PBS to get rid of disturbing molecules, like glycerol and sodium azide. I use Slide-A-Lyzer™ MINI Dialysis Devices, that works perfectly for me.

good luck

Rene

Thanks Rene, that's really helpful. We might give that a go next!

Hi Tabitha,

The Slide-A-Lyzer mini dialysis device is an efficient & simple way to remove the presumptively offending glycerol.  Good luck, Tabitha.

Bruce

Hi Bruce,

Thanks for your reply, sounds like the Slide-A-Lyser is the way to go!

considering thatyu have aflow cytometer, the best way to check the quality of your bead conjugation is to stain them with anti-species antibody. I use Goat-anti-mouse Fab2xPE from Dako

http://www.wiley.com/legacy/products/subject/life/cytometry/isac2000/6579.htm