thank you john for your reply.

Sure you can! In fact, there is no mechanistic difference between IP and co-IP. I feel from your question that it might help you if I elaborate this subject a bit--never mind if you already know the following.

When you immunoprecipitate your protein of interest 1 (YPI1) and examine itself, it is called IP. When you immunoprecipitate the same YPI1, but examine whether YPI2 or YPI3 come along, it is co-IP. Thus, in actuality, you do not have to use any special kit. All you need is a good general-purpose cell-lysis buffer (e.g., comprising Tris-Cl or Na/K-Hepes, ~20mM, pH ~7.5; salt NaCl/KCl around 150mM, EDTA ~1mM, some MgCl2 1-2mM; 5-20% glycerol, a detergent like Igepal/Triton at 0.2-1.0% and protease inhibitors). This is a standard buffer for most soluble proteins, and is fairly compatible with antibody-antigen interaction. You can adopt various easy and inexpensive cell lysis methods. The buffer condition may need tweaking in special cases (e.g., if YPI1 is tightly bound to chromatin).

That said, there is a fundamental difference between IP and co-IP that you need to keep in mind. IP simply involves lysis of of the cell into a soluble soup of proteins where the antobody will bind its specific antigen. Since the antibody-antigen interaction is stable in a wide range of conditions in ionic strength, pH and detergent concentration, you can have the liberty of using harsher or more-stringent buffer conditions in order to get a very clean IP. But in co-IP, you need to maintain a condition where the interaction of YPI1 with YPI2, YPI3 etc remains unperturbed. That might require some trials until you find the best conditions to suit your targets. Therefore, if you do not see Western blot signal for YPI2 in YPI1 IP, it does not necessarily mean that YPI2 and YPI1 do not interact inside the cell. It simply means that they did not interact in the "cell-free" conditions that you provided. The other factor that might negatively affect a co-IP experiment maybe beyond your control: that is, if the Ab and YPI2 recognize the same region on YPI1.

Thanks Anil for all the information you have provided.

Hi, 

The examination of protein-protein interactions in the form of co-immunoprecipitation can involve very weak and short lived interactions, such as the detection of adaptor proteins binding to phosphorylated tyrosine or ser/thr residues on the cytosolic tail of activated receptors. Alternatively, the detection of all binding partners to a certain protein (possibly acting as hubs) demonstrated by 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. Fascinating work was performed by the group of Albert et al. 2007 (Nature 450, 683-694) examining nuclear pore complexes and interactions using downstream mass spec. This group used antibody covalent coupled to Dynabeads M-270 Epoxy (these beads have very low background to avoid contamination of the preparation). They did spend a significant amount of time to find the correct buffer composition to capture these weak and short lived interactions - buffers which are part of the Dynabeads Co-IP kit. As every interaction is different they need to be optimized individually. The 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.

Components that can be varied to increase or decrease stringency of the Co-IP include;

• Salts

• Detergents

• Dithiothreitol (DTT)

• Protease Inhibitors

Buffer modifiers

• Sodium Chloride (e.g., 1 M NaCl)

• Dithiothreitol (e.g., 1 M DTT)

• Magnesium Chloride (e.g., 1 M MgCl2)

• Potassium Acetate (e.g., 1 M KOAc)

• Tween-20

• Triton X-100

• Protease inhibitors without EDTA

In terms of Ab coupling to the beads - up to 10µg one will find a linear relationship between added Ab and coupled Ab. Above these concentrations the Abs will precipitate down to the bead surface but a reduction in covalent bonds is seen. Actually, less than 10 µg ensures efficient covalent binding (no leakage) and functionality. coupling of large amounts of Abs (20-30microG Ab/mg beads) is not recommended. If loading 20µg of Abs to the beads the total amount of Abs covalently coupled to the beads will not be more than 13µg/mg beads.

kind regards

ketil