Mick.Partis at BBSRC.AC.UK
Thu Nov 23 09:08:33 EST 1995
Philippe Pognonec <pognonec at unice.fr> wrote:
>I have a protein (pI:+/- 5.3), and I have an epoxy-activated agarose.
>But I don't have a GOOD procedure to effectively put them together.
>Is there anybody around here who could help me on that matter?
>Thanx in advance!
I have found that the procedure given by Pharmacia for use with their
epoxy-activated Sepharose works well.
Re-swell freeze-dried epoxy-activated agarose with about 100ml distilled
water/g agarose. 1g freeze-dried material gives approx. 3ml gel. Wash
the gel in coupling buffer (any buffer with a pH between 9 and 13, which
contains no nucleophiles such as Tris or glycine, will do: I usually use
carbonate or phosphate).
Dissolve your protein in the same coupling buffer and incubate with the
agarose in a shaking water bath at 30C for 16 hours.
Wash off excess ligand with coupling solution, followed by distilled
water, 0.1M bicarbonate, pH 8, and finally 0.1M acetate buffer pH 4.
Block with 1M ethanolamine overnight, wash off excess ethanolamine
with 0.1M acetate, 0.5M NaCl, followed by 0.1M borate, 0.5M NaCl, and
store at 4C.
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