Could be incomplete reduction or impurities. Have you tried DTT or TCEP instead of BME? Do you see the same pattern when you run a Western and probe it with a polyclonal anti-IgG?

Hi, Holly,

I think the best conclusion will be brought by doing a Western blot. Then it can be determined where is each of the heavy and light chains. Pay attention to complete reduction and denaturation before the SDS PAGE. If partial reduction occurs, bands can show unexpected sizes.

Amal.

Hello, Holly!

I used to purify bulk prep myeloma protein and monoclonal antibody in the 100 milligram to 1 gram range.

The most likely contaminant in the 65-70 kD range is serum albumin. Virtually every method of antibody purification [SAS cut, protein A or G column, even affinity/anti Ig columns] will contain some. Typically, we note it and ignore it.

If you are purifying antibody from a hybridoma, the small band in the13-15 kD is likely light chain fragment from the myeloma fusion partner. It is the result of an aberrant VJ joining event that results in the production of a kappa light chain with no variable region. The original myeloma that the fusion partner was generated from has this fragment, and all hybridomas produced by it have this fragment. Again, it is noted and mostly ignored.

Gary