::Here I am telling you that Periodate method is one of the most
:inefficient coupling procedures, particularly for the protein sample
:with low concentration.
Did you try "my" protocol? :-) How can you tell? But, yes, the coupling
efficiency is highly dependent on protein concentration. For most
proteins, around 2-3 mg/ml (final after 1:1 dilution with sepharose
suspension) is optimal.
Exactly because of it's "unefficiency", it is the most gentle, preserving
protein activity MUCH better than most others.
:I am suggesting some of the direct, one-step and simple but highly
:efficient activated matrices comercialy available.
None of them is more one-step than periodate. That is, if you don't
consider activation and buying a "non-step". Plain CL Sepharose
is dirt cheap (because it it sitting unusable for decades in every
cold room without exception), pre-activated matrix is very
expensive.
:1. Activated ECH Agarose. Available from both Peirce and Sigma.
:Providing 6-carbon spacer and an active NHS ester as leaving group and
:form stable amide linkage with your amine containing protein.
:2. Cynogen Bromide activated Sepharose. One of the most efficient
:matrix available and provide one carbon spacer but has very bad
:reputation for leaking and ionic exchange effects.
:3. A novel activated matrix developed by 3M and sold through Peirce
:Chemical. The matrix contains a active lactone, which is readily form a
:stable amide with primary amines of the protein. Disavantage: hyarolysed
:lactone create a free carboxyl=ionic effect; low flow rate of its
:acrylamide polymer matrix.
:4. Activated thiol Agarose. Available from Sigma. It forms -S-S-
:bond with free thiol (cysteine residue) of the protein effiently with
:high yield (the matrix is not sensitive to water as others).
:Disavantage: you need to reduce your protein with DTT first before
:coupling.
:5. Iodoactate activated Agarose. Available from Peirce. High yield
:and stable for free thiol containing protein.
I listed these myself in this very same thread previously :-)
I only did not list thiol-based because, in my opinion, they are
unsuitable for any serious application involving crude material.
Oxyrane activated Sepharose (Sigma, I thinlk), is also worth
consideration
:The disavantage of Periodate activated Agarose:
:need high pH (>pH9) to
:obtain a good high yield and efficient;
9.0 is good enough. Almost never a problem for most proteins
(unlike pH < 5 which kills a lot of them).
:Almost no good for protein less
:than 0.1 mg/mL.
True. I wouldn't even try it with 0.5 mg/ml. To various extent,
the same is true for all listed above.
:The formation of Shif-base is not reversible unless
:furthewr reduced to secondary amine (harmful to lots of proteins).
???
Did you read my post? The reduction is indeed used, creating
irreversible linkage. No, I don't think borohydrid used as described
is so harmful. I know from my own experience IgG and IgM sorbents
prepared in this way perform much better than others, and I was told
that several immobilized enzymes had higher activity too.
:Oxidation damage the matrix and create leaking problems.
There is no any kind of "damage" I am aware of under this procedure.
Any serious leaking was not observed. In fact, it was _much_ less than
for NHS and cyanogene-based sorbents.
:Beside, I have to warn the one who put the first question: affinity
:purification of antibody using whole protein will create lots of
:problems and limits the application of the antibodies because of the
:leaking of antigen, WHICH CONTAINS MULTI-EPIOPES. Unlike eptope-based
:affinity chromatography, the leaking antigen will interfere the
:interpretation of your results.
While this can be a concern for industrial applications, it has no
importance at all in research. First, the amount leaking is very small in
all reasonable cases, second, all intelligent people should do controls
anyway (mock or non immune pturification over the same column).
For what it is worth... I only speak from my own experience. Nothing
is "the best" in all cases, and every particular application plus
common sense and knowledge dictates the choice of sorbent.
Regards,
- Dima
