Ab purification problem
dk at no.email.thankstospam.net
Thu Oct 17 21:16:47 EST 2002
ljd3 at duke.edu (Lou D'Amico) wrote:
>i used a 3mg/ml (little less) solution, i had about a (very) little
>over 1ml of that, and i bound it to 1ml of column matrix. It's the
>same volume as the company i outsourced the first affinity
>purification to. Any help?
Some. It would help if you could state how much of your antigen is
bound to the matrix, not just how much you used. Depending
on many factors coupling efficiency can vary greatly from
as low as 10% to nearly 100. Still, lets do some rough numbers:
Assume the antigen is actually coupled. Say, 75% of it, 50% in
the worst scenario. So you have ~1 ml of ~ 2 mg/ml column.
Because it's serum, it is reasonable to expect more than 1:1
molar binding. Unlikely more than 3 because of steric
hindrances, etc. OK, so that means you can expect at least
2/2.5 mg Ab = 800 ug (150K = 2.5x60K). You say you actually
got 500 ug, which is not far off. In ideal world I would expect
about 3X of this. But since you did not select for IgG first, it
is quite possible that i) serum was collected prematurely and
has a lot of IgM ab, ii) you failed to elute those IgM (they are
harder to elutel; you don't mention how you elute and if you
checked on a gel what remains bound). All in all, your result does
not seem to be impossibly wrong.
Good antiserum can contain between 0.05 to 0.2 mg
of specific antibodies (speaking of rabbits at least). So your
Western, which is, of course, non-quantitative, should indeed
show that the serum is not yet depleted.
In times when I had only a little of antigen that is too hard to
come by, I used to do this:
- Purify IgG on Protein A, elute with 100 mM Glycin, pH 2.5,
immediately neutralize to pH 8.0 with 2 M Tris, pH 9.0,
add NaCl to 0.35 M final.
- Run over an antigen column, load 2 times to ensure saturation
of binding sites, with 3.6 M MgCl2, pH 6.5. Dialyse extensively,
make sure to leave room for water in dialysis bag. MgCl2
elution is a lot milder on most native proteins than standard
Hope this helps.
>On Thu, 17 Oct 2002 03:08:00 GMT, dk at no.email.thankstospam.net (D.K.)
>>ljd3 at duke.edu (Lou D'Amico) wrote:
>>>I'm trying to affinity purify an AB i generated in guinea pig to an
>>>insect enzyme (JH-esterase). It's approximately 60kD. When we had a
>>>company originally do the affinity purification, the yield was low
>>>(maybe 500 ug out of 20mls of serum). The serum that passed through
>>>the column was still chock full of reactive Ab (confirmed by Western
>>>Blot). I did another purification in house on an IgG purified extract
>>>of the serum, and still got low yields in the purification (maybe
>>>300ug). I don't have tons of the antigen to work with, and I was
>>>curious if someone could help me with two questions.
>>>1) Does the size of the antigen impact yield this severly? I was
>>>coupling 2-3mg on a ml of column matrix.
>>>2) Are there standard alternative techniques to purifying Ab's of
>>>large mol weight proteins that might be more appropriate?
>>3 mg/ml bound protein is typically optimal for affinity
>>chromatography. What was the volume of column used in
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