8 replies to this topic

[Mighty Mouse]

I was just curious if anyone has experienced any differences between using Protein A or Protein G for ChIP'ing antibodies of rabbit origin? The little literature I've been able to find suggests that either it doesn't matter if one using Protein A or G for rabbit antibodies or that Protein A would bind better to rabbit antibodies than protein G.

Thanks

MM

[aimikins]

it's my understanding that it depends on isotype, not species origin, but I may be wrong here.

[Mighty Mouse]

You may be right, however, everywhere I've looked up the affinity of Protein A/G for various antibody types they lump together Rabbit IgG into one category, whereas they have various isotypes of Mouse and Human IgG. I'm no immunologist by any means, so I'm not altogether sure what this implies in terms of Rabbit IgG isotypes...

[epi123]

my understanding is that protein A beads are generally better suited for rabbit polyclonal antibodies. i've seen the mouse monoclonals enrich better with protein G. Years ago i would use protien a/g sepharose beads but lately i'm using the MAGnify kit from invitrogen that uses a mix of protein a/g magnetic beads

[Mighty Mouse]

Thanks for your response epi. I had originally been working with protein G beads and using a mouse antibody to test out ChIP, but am moving into using some rabbit antibodies and have had some difficulty getting decent enrichment with the protein G. I'm hoping by switching to protein A magnetic beads this will clear up the issue.

MM

[jhb80]

This one might be a useful table (from the Invitrogen website) - doesn't quite solve the rabbit isotype issue, but they are typically polyclonal anyway...?

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[Mighty Mouse]

Thanks, I've seen this from invitrogen. However, I've seen some other stuff (e.g, the book: Using Antibodies: A laboratory manual by Ed Harlow and David Lane) that suggests one should use protein A for rabbit antibodies during immunoprecipitation. Hence my confusion I suppose, different sources have different suggestions and I was wondering if anyone had any experience with this issue.

If I get any noticeable differences with using protein A for protein G beads I'll be sure to post for anyone else that may have this question.

MM

[giny]

Thanks, I've seen this from invitrogen. However, I've seen some other stuff (e.g, the book: Using Antibodies: A laboratory manual by Ed Harlow and David Lane) that suggests one should use protein A for rabbit antibodies during immunoprecipitation. Hence my confusion I suppose, different sources have different suggestions and I was wondering if anyone had any experience with this issue.

If I get any noticeable differences with using protein A for protein G beads I'll be sure to post for anyone else that may have this question.

MM

MM, can I know from where you get your protein A beads from? i would like to try using protein A beads too since i dont get much DNA enrichment after using Protein G beads. Thank you very much

[Mighty Mouse]

Sure thing...I get them from Millipore. I've been using their Magna ChIP kits, although I believe they sell their magnetic beads separately. Just as a follow-up to my previous post, I'm still not sure if I'm seeing a difference with the protein A beads yet...