A problem with IgG purification by Protein G chromatography
Posted 19 October 2011 - 05:47 AM
I have a problem with my purification of mouse antibodies by Protein G affinity columns. I have several hybridomas and I can purify the corresponding antibodies with fairly good yield....in all cases except for one. In principle, the problematic hybridoma could produce the antibody at low levels.
However, it calls my attention the fact that all the good antibodies have kappa light chains whereas the problematic one is lambda.
Theoretically, protein G binds the Fc of the antibody. Consequently, the light chain isotype shouldn´t affect the binding. However, I trust your experience -guys- more than books!
Could anyone confirm whether there could be a difference in binding capacity of Protein G depending of the light chain isotype?
Thank you in advance!
Posted 19 October 2011 - 09:49 AM
Posted 19 October 2011 - 10:11 PM
I would not recommend protein A for this because protein A binds most mouse IgGs with very low affinity. If you want to purify mouse IgG1 with protein A you have to use a very high concentration of salt in your buffer.
What I would do first is to check the production yield of your cell line in case it's not producing well and your purification is OK. Are you sure of the isotype/subtype of your Abs. For example Abs of the IgM class don't bind protein G.
Posted 20 October 2011 - 05:05 AM
Posted 24 October 2011 - 09:03 PM