Basic De52 Ionexchange Chromatography With Triscl Protocol

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DE52 ion-exchange (IEX) chromatography can be used to purify antibodies from a tissue culture supernatant, ascites fluid, and serum or ammonium sulfate precipitates derived from any of these antibody-containing fluids. The protocol may also be used as a second step following purification by size exclusion (SE) chromatography (see Basic Protocol 1 and Alternate Protocols 1 and 2). The major contaminant protein in all these preparations is albumin, which binds DE52 tightly under conditions of low-to-moderate ionic strength. Antibody either fails to bind to DE52, in which case it elutes in the void volume as the column is loaded, or it binds loosely, and can be eluted with a gentle salt or pH gradient. Fractions eluted are assayed first by A280 and then by either ELISA (unit2.i) or SDS-PAGE

Caution should be exercised in that different monoclonal antibodies as well as antibody fractions from different immunized animals' sera elute from DE52 under different conditions. In this protocol, antibody in 0.01 M TrisCl at pH 8.6 is passed over the DE52 column and bound antibody is eluted with a gradient in the same buffer approaching 0.5 M NaCl.

Materials

DE52 powder (Whatman)

Antibody sample (ascites fluid, tissue culture supernatant, immune serum, or ammonium sulfate precipitate) 1.5 x 50-cm column

Additional reagents and equipment for column chromatography (appendix 3i), dialysis (appendix3h), and ELISA (unit2.1) or SDS-PAGE (unit8.4)

1. Swell DE52 powder in 0.01 M TrisCl, pH 8.6, and remove fine particles.

It is very important to adjust the pH prior to pouring the DE52 gel into the column.

2. Pour a 1.5 x 50-cm DE52 column and equilibrate with 0.01 M TrisCl, pH 8.6.

The size of the column is dependent upon the total amount of protein in the crude antibody sample. The capacity of DE52 is >100 mg total protein/ml hydrated gel, so a column of 1 to 5 ml is commonly sufficient.

3. Place antibody sample in dialysis tubing. Dialyze twice against >20 times the sample volume of 0.01 M TrisCl, pH 8.6, overnight at 4°C.

Purification of IgG

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  • isembold
    What is DE52 chromatography?
    8 years ago

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