4 replies to this topic

[Chelo]

Hello everyone,

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!

[LifeTein Peptide]

You can try to use the protein A/G columns. In case the antibody could not be purified by protein G, you still get a chance to have protein A for it.

http://www.piercenet...14-291106931E06

[BioMiha]

The light chain is not a factor for several reasons. 1. because as you said protein G does not bind the light chain and 2. because the majority of mouse light chains (~95%) are kappa, so yours is probably as well.
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.

[LifeTein Peptide]

BioMiha made good points. Protein A/G from Pierce is not a mixture of Protein A and Protein G. The monoclonal IgG antibodies subclass identities have not been determined. That is the reason I recommend the Protein A/G column from Pierce because it binds to all human IgG subclasses. Protein A/G binds well to all mouse IgG subclasses but does not bind mouse IgA, IgM or serum albumin. This makes Protein A/G an excellent tool for purification and detection of mouse monoclonal antibodies from IgG subclasses, without interference from IgA, IgM and murine serum albumin. Individual subclasses of mouse monoclonals are likely to have a stronger affinity to the chimeric Protein A/G than to either Protein A or Protein G.

[rick112]

yup, there could be affinity difference between various antibodies towards Protein-G, i also think protein A/G column suggested by 'LifeTein Peptide' will be a better option when dealing with array of monoclonal antibodies