Protein immobilization on Sepharose using periodate activation (Was: Purifying Antibodies using affinity support)
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Tue Mar 9 17:05:15 EST 1999
In article <7c3oi7$rhj$1 at mserv2.dl.ac.uk>,
wolfgang.schechinger at med.uni-tuebingen.de wrote:
:Suppose the periodate protocol is oxidizing cis diols into
:bis aldehydes and reduce the imines formed after coupling with
:cyanoborohydride to amines. And clening leftover CHOs with boranate.
:But this procedure above probably might kill any active enzyme. Is
:there a way to prevent this?
:You remember the exact protocol by chance?
Here you go. I found the old file and tried to make it look like a
PROTEIN IMMOBILIZATION ON CROSS-LINKED SEPHAROSE
USING PERIODATE ACTIVATION
1. Wash Sepharose CL-4B (not cross-linked version DOES NOT
work!) with 10-20 volumes of pure water on glass filter.
2. Mix one volume of packed Sepharose with two volumes of
20 mg/ml sodium periodate (NaJO4) in water, incubate for
two hours at room temperature on rotator.
3. Wash with 5 volumes of water, then at least 3 volumes
of 100 mM sodium borate, pH 9.0-9.4 (pH is dictated by
your protein stability; at 9.4 the coupling proceeds more
efficiently, but still quite OK at 9.0) on glass filter.
(This gives you activated Sepharose; at this point it is
quite stable and can be stored at +4 for at least one month).
4. To one volume of packed Sepharose, add 1-3 volumes of
protein solution at 2-5 mg/ml in the buffer with pH 9.0-9.4
that is free of amines. (For example, one volume of 5 mg/ml
IgG in 200 mM sodium borate or bicarbonate, pH 9.4).
Incubate on rotator at rt for three hours or in cold room
5. Sediment, collect supernatant, save, wash 2X with two
volumes of 100 mM sodium borate pH 8.0 by resuspension/
sedimentation, collect washes. (Determine coupling
efficiency after measuring protein concentration in
original super and washes; usually around 80% bound).
6. To the Sepharose, add equal volume of FRESHLY prepared
3 mg/ml sodium borohydride (NaBH4) in 100 mM sodium borate,
pH 8.0, incubate on rotator for 5 min, wash repeatedly
with sodiun borate pH 8.0 until pH in the super is ~ 8.0
(use pH paper).
7. For the paranoid: remove non-specifically bound protein
and block residual reactive groups by washing 3X with 2
volumes of 100 mM ethanolamine, 0.5 M NaCl, pH 8.0
8. Wash with buffer of your choice, add azide to 0.01% and
store at +4C. Alternatively, add equal volume of ethylene
glycol or glycerol, allow to mix throughly on rotator and
store at -20C.
Sorry, no reference; I know for a fact that the person who
developed this method somewhere around 1985 is Dr. V.V.Sinitsyn,
then in the Institute of Experimental Cardiology, USSR Academy of
Medical Sciences, Moscow; I could not find any published
reference to this method and the above is what I used personally
and can attest to be working. PLEASE, if using the method,
find some way to acknowledge the author and the source. I am
afraid he is no longer working in science (thanks to the
revolutionary reforms in Russia that left no way for most scientists
to make a living). I am leaving "X-No-Archive: yes" out to get
this post archived in Dejanews, so I imagine referencing the
post by providing URL would be OK.
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