Concentration and conjugation of antibodies

Guy Hermans guy.hermans at
Fri Nov 18 13:43:32 EST 2005

Hi Marianne,

You didn't state which volume you started off with before the gel filtration
column -assuming it got more than 1.5x diluted, this can be helped by using
a shorter, narrow bore column. It depends on the Sephadex you're using, but
usually resolution is so good with small MW dyes you don't need very long
columns. Using a narrow column also helps to reduce the dilution effect.

You can also remove unreacted dye using dialysis, which cuts down on the
dilution but takes time.

Using centricons can also be used to both remove unbound dye and concentrate
the material; it also takes less time than dialysis, but occasionally all
material gets lost (I suspect binding to the plstic/membrane). Use similar
cutoff filters as for dialysis: 10k or should work just fine for small MW
dyes like Cy.

Lastly, for many applications you don't even need to remove the unbound dye,
as you're washing off unbound antibodies etc. after staining the sample.
Just be sure you've added enough decoy to quench all remaining reactive
molecules or you'll end labeling your sample - Tris or ethanolamine do
nicely with succinimidyl dyes.


-----Original Message-----
From: Marianne.Enger at [mailto:Marianne.Enger at]
Sent: Friday, November 18, 2005 3:47 PM
To: cyto-inbox
Subject: Concentration and conjugation of antibodies

Dear Flowers;
I have a postdoc in my group who wants to conjugate antibodies and he has
asked me to post this question:
I want to conjugate antibodies (1 mg/ml) with Cy5 dye, however I am un sure
to the best way to concentrate the conjugated dye after separation on
column. In previous attempts, I have gotten about 6 ml of conjugate back
the sephadex column following separation from unconjugated dye...and the
resulting solution is not stable. Does anyone have any suggestions on
concentration procedures to get a final antibody-conjugate solution of 1

Emmet McCormack

Marianne Enger
Dept of Internal Medicine
Hematology section
University of Bergen
Haukeland University Hospital
mail: marianne.enger at

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