What type of Dynabeads were you using? They sell 2=A1 Ab coated beads tha=
t should=20
isolate your polyclonal and/or monoclonals adequately. I noticed anti-mou=
se,=20
rabbit, and rat, as well as specific anti-mouse subtypes.=20
We've used the tosyl-activated beads to immobilize peptides for mini affi=
nity=20
chromatography columns. We double-coupled a 1-2 fold xs of peptide overni=
ght at=20
room temperature using 2ml tubes with about 1.5ml of solution. We found t=
he=20
amount of liquid in the tubes as well as the tube size played a large rol=
e in=20
getting peptide attached. We probably would have been better off using mo=
re=20
peptide, but some samples were too precious. We followed the peptide trea=
tment=20
with a 4h incubation with 1M tris to block any extra tosyl groups, then w=
ashed=20
3x with PBS and stored the beads in PBS until use.=20
We tested the beads by AAA for peptide content and were pleased with the=20
results. 50=B5l of beads was sufficient to isolate a reasonable amount of=
protein=20
from a whole cell lysate. Our yields from the beads allowed for identific=
ation=20
of that protein by repetitive IPs. You should have much better luck using=
an=20
antibody followed by MS due to the difference the binding affinity and=20
detection.=20
In my experience these beads were easier to use than sepharose or agarose=
beads.
They do cause a bit more non-specific binding than sepharose, but that's =
easily=20
taken care of by a short pre-clear step with tris-blocked beads. I've nev=
er had=20
good luck with agarose beads. Maybe I just don't have the touch, but it s=
eems=20
that I either isolate a bunch of garbage or nothing at all.
If you have any questions, let me know. I can send you a copies of our ac=
tual=20
protocols if you think it would be helpful.
Jeni
Janelle Lauer-Fields
Department of Chemistry & Biochemistry
Florida Atlantic University
777 Glades Road
Boca Raton, Florida 33431
561-297-2094/Fax 561-297-2759