If you have the antibody bound to a column, you could try electroelution of the protein from the gel. This method is used for affinity-purification of immunoglobulins, to avoid getting mediocre antibodies (poor antibodies do not bind to the column, high affinity ones are not eluted). It also avoids the use of protein denaturants.

Engelbert Buxbaum Thanks for answering; the beads are magnetic so electroelution may not be an option. I also would like to capture all antibodies (including poor/high affinity ones) as I am analyzing an antigen-specific IgG repertoire.

Boiling the beads in SDS-PAGE sample buffer should elute everything. If not, try including 6M urea in the sample buffer. Since reducing conditions are used, each IgG will appear as 2 bands (heavy and light chain), but since there are many different antibodies in an antiserum, all slightly different and with varying degrees of glycosylation, you will actually see broad bands centered at about 50 and 25 kDa.

Hi Robert,

There are many things that can be used to disrupt the binding of the antibody to antigen on the beads, you have already used SDS, but you could also try other approaches such as high/low pH or use of chaetropic ions. Some examples would be:

(I would try pH first, less chance of irreversible denaturation). The list below are the extremes, if you find one that works but you get irreversible denaturation, try going to a less extreme value and see if you can get a reasonable mix of recovery and activity.

You can find a better explanation at:

Using Antibodies: A Laboratory Manual (Harlow and Lane, second edition, September 2013)

Chaetropic ions

Urea (up to 6M)

NaSCN (up to 3.5M)

pH

Low pH:

• 100 mM glycine pH2.2-2.8
• 100 mM citric acid buffer pH 3.5-4

High pH:

• 100 mM glycine-NaOH pH 10.5
• 0.05 mM diethylamine pH 11.5
• Sodium borate pH 10