Antibody coupling to Magnetic Beads - (Jun/23/2009 )
Thought I would ask in the immunology section, please let me know if this topic might be appropriate elsewhere.
We have an antibody which is great for IP, only thing is it is only supplied in 50% glycerol. We want to couple/conjugate to magnetic beads so that we can reuse the antibodies and elute off only our antigen.
1. I tried using crosslinkers (DMP) which utterly failed, the binding of antibody to antigen was less than that compared to free antibody.
2. We want to use Tosyl-activated Dynabeads from Invitrogen, but according to Invitrogen, glycerol will compete with the tosyl-amine reaction. How can we overcome this?
3. Is it possible to have a little bit of glycerol lying around? perhaps I can dilute our antibody to say 5%. The worst is I might need dialyze glycerol. Has anyone ever dialyzed a small volume containing 50% glycerol? I don't know where to start and we can afford to lose antibody.
Please help, I am lost and reference books do not discuss such trivial issues (which it is not, it's really annoying actually). Any help would be appreciated!
I think your ab is the primary concern. If Dynabeds and invitrogen can not help you maybe you can ask Sphereotec or Seradyn or Bangs labs for assistance. They all make magnetic particles and have technical assistance available.
I think you should be able to dialyze out the glyerol. Pierce makes device for dialysis of small volumes. If you ab is concentrate you could dilute slightly.
the problems you have are because you want to bind the antibody to something that will give a random orientation of the ab. that's why the crosslinking reduced the apparent avidity.
the tosyl-activated beads will bind the ab in a random orientation. this will lead to the same problem. and it will be interfered with by the glycerol.
you can bind the antibody to protein a or g and crosslink with dss (thermo pierce has kits for this: see this and this). you can bind protein a and/or g to magnetic beads and then bind and crosslink the ab.
as for dialysis, what volume do you want to dialyze?
you can dialyze droplets by floating a piece of dialysis membrane on top of buffer then place a droplet onto the membrane.
you can use narrow dialysis tubing to dialyze samples of less than a ml.
or you can use a slide-a-lyzer from pierce. see this page.
The surface of Dynabeads M-280 Tosylactivated is slightly hydrophobic and will therefore favor binding of the Fc part of the antibody which is more hydrophobic than the Fab part. The orientation of the antibody bound to the beads will therefore be predominantly with the Fab part facing outwards, available for binding to it's target.
We also have Dynabeads M-270 Epoxy, which are uncharged and exhibit random orientational binding and extremely low background binding.
Preservatives like glycerol will react with the Tosyl groups on the beads, it is crucial that glycerol or other preservatives are removed from the antibody before coupling. Several vendors provide devises for dialysis of small volumes. My favorite is the Slide-A-Lyzer Mini Dialysis Unit from Pierce. http://www.piercenet.com/products/browse.cfm?fldID=04010165 Recovery should be >90%.
The antibody will be covalently coupled to the beads, so if you use mild elution conditions after target capture, e.g. 50 mM Glycine pH 2.7-3, then the beads can be washed and reused several times. You should be aware however, that as elution is never 100% efficient, some target protein may be left on the beads after elution. Therefore, it is recommended to re-use beads only for similar samples or for samples where carry-over will not matter.
Antibody coupled Dynabeads M-280 Tosylactivated will typically bind 1-10 µg of target protein.
I hope this helps! Let me know if you have any further questions. You can also write to us at firstname.lastname@example.org with 'Dynabeads' in the subject line. We try to answer all our emails the same day.