Summary: Auble Lab
Purification of EE-Mot1 Using Antibody-Coupled Beads
Perform all steps except quick spins and peptide elution at 4 degrees. Use about 100 mg of
yeast extract protein per binding reaction. Dilute extract in Benoit's buffer 1:1 with HEG + 0
containing DTT and protease inhibitors. (Add the HEG + 0 slowly to the extract with
Save 6 µl diluted extract for western blot.
Wash 0.5 ml Py antibody-coupled beads with 10 ml HEG + 0.3 M KOAc containing 1 mM
DTT, protease inhibitors and 0.1% octyl glucoside.
Set up binding in conical tube by adding 0.5 ml pre-equilibrated Py antibody-coupled beads
to diluted extract.
Incubate binding reactions for 2 hours at 4 degrees with gentle mixing.
Spin binding reactions for 2 minutes at 4 K rpm in clinical centrifuge. Remove supt and
wash beads with 3 X 10 ml HEG + 0.5 M KOAc plus DTT, protease inhibitors and 0.1%
NP40. Save 6 µl unbound supt (first spin) for western blot.
Wash beads once in 10 ml Tony buffer containing DTT, PMSF and Benzamidine.
Elute bound Mot1 in 2 X 1 ml Tony buffer containing 200 µg EE peptide per ml by
incubation of the beads at room temperature for 10 minutes for each elution. Save 6 µl of
each elution plus 6 µl beads for western blot. Store at 80 C and check purification by