Hi Chris, 

May I ask if you refer to the Dynabeads Co-IP kit for covalent coupling of primary Abs (includes buffer for Ab coupling and buffers for optimizing the conditions for protein-protein interactions) or is it the Dynabeads Protein A or G beads? 

kind regards

ketil

Hi Dr. Pedersen,

It was either kit really. I am looking to perform a Co-IP with my poly-clonal antibody, but either kit should work with me. But I think what you have said already answers my question. Covalently linking my Prim Ab to a bead is going to cost more than linking it to the protein A or G and performing the experiment that way. Is that correct?

Hi Dr. Brigolin,

In terms of covalent binding this will cost more than stand-alone Dynabeads protein A or G beads as it also contains 2 sets of buffers, one for coupling and one for optimizing the co-IP conditions. its also different surface chemistry of course - so a different magnetic bead. So I guess it all boils down to whether non-covalent binding of prim Ab is sufficient or not as the Abs might be eluted off together with the target(s).

Other things to considern when selecting the tools to use is:

- the background or unpecific binding in order to have the best signal/noise

- antibody binding capacity (which will depend on the Ab class of choice, and the affinity in order to avoid Ab leakage during bead preparation and prior to target incubation which would of course give low yield and be expensive).

- yield (amount of target(s) isolated.

-reproducibility

- protocol (ease of use)

- incubation times

- price/test

the choice might also be reflected in the type of co-IP performed. Protein-protein interactions often involves very short lived and weak interactions, such as adaptor proteins binding to phosphorylated Tyrosine residues on the cytosolic tail of activated receptors - which I used to work with studying the Epidermal Growth Factor Receptor and adaptor interactions with the cytosolic tail due to EGF stimulation. Or if one wants to detect all molecules binding to a certain protein such as the very interesting study of Praefke et al 2004 who identified binding partners to adaptor protein 2 (AP-2), PIP2 and clathrin (Praefcke et al The EMBO Journal VOL 23 | NO 22 | 2004. Very interesting work was also done by the group of Albert et al. 2007 (Nature 450, 683-694) who studied nuclear pore complexes and interactions followed by downstream mass spec. This group used the Dynabeads M-270 Epoxy in combination with optimized buffers as these beads have very low background to avoid contaminate the preparation. They did spend a significant amount of time to find the correct buffer composition to capture these weak interactions. Actually, these buffers are part of our Dynabeads Co-IP kit which includes Dynabeads M-270 Epoxy and Ab coupling buffers in addition to co-IP buffers. These buffers offer the opportunity to optimize and fine tune weak and transient interactions between short amino acid sequences in different proteins in terms of stringency.

feel free to contact me at any time.

kind regards

ketil

That is great information. Thank you very much!

Its all my pleasure. Feel free to contact me at ketil.pedersen@thermofisher,com as well.

good luck

ketil