In the past several years I am doing IP-WB, but so far there is no consistent and standard protocol to work on all IP's. So, here my question is I need very detail and specific steps need to consider to get good result in IP's. Need suggestions in the following specific steps,
1. IP-lysis buffer
2. Washing beads
3. pre-clearing step
4. Elution step
Thank you for your valuable time spent on this issue...
I highly recommend the resources on the Thermo website. Here's a link to get you started. Good luck!
http://www.piercenet.com/method/immunoprecipitation
Hi Beverley Dancy,
Thank you, i go though your attachment. I got clear most of the aspects...
Thanks
you can also use the MILLIPORE Catch and release IP kit. It is worth it. My personal recommendations for this KIT.
quality of elutions is very pure and clear when u monitor on gel.
Hi Sabyasachi Dash,
Thank you for you suggestion, i will try Millipore kit...
Hi Inga Koneczny,
so for in our laboratory we didn't start to use magnetic beads, I talked to my PI regarding this issues, soon we are going to start magnetic beads IP-WB. Thanks for your detail comments and suggestions...
There is no general protocol that works for every situation. There are many many considerations, here are a few:
1) Are you doing an IP or an co-IP? For a straight IP (for example followed by MS identification of phosphorylation sites) then agarose beads are better. There is less background with agarose beads and their binding capacity is much much higher. Agarose beads will typically have a binding capacity ~0.5 - 1.0mg/50uL beads. The binding capacity of magnetic beads is typically on the order of 4-6ug per 50uL beads.
2) Magnetic beads however are preferred for IP of large macromolecular complexes. This is because the agarose beads are porous and much of the antibody actually sits inside of the bead rather than on the surface of the bead. The small pore entrances can occlude the entry of large macromoleuclar complexes. Magnetic beads are not porous therefore all of the antibody sits on the surface of the bead allowing for the binding of large complexes.
3) What is the isotype of your antibody? Depending on the isotype you'll need to use protein A, G, AG or protein L. In the case of magnetic beads you can also use anti-mouse or anti-rabbit beads etc. However note that the optimal pH for antibody conjugation varies for each protein type. For protein G the antibody binding is optimized around pH 5.2-5.5 while it's ~8 for protein A.
4) Blocking beads; if you use magnetic beads you should really consider blocking your beads with IgG-free BSA (5%)
5) what is the downstream application? i.e. should you covalently or non-covalently conjugate your antibody of interest.
6) Lysis buffer: we all tend to use NaCl but this is actually quite different than the intracellular electrolytic profile of a cell. Some proteins require high K to maintain their conformation. Consider a 150mM K rather than Na buffer. Other proteins might preferentially interact at a particular pH.
7) What other factors might affect your protein-protein interaction for co-IPs. If you're trying to co-IP a kinase and it's substrate consider adding Mg and ATP. If you're interested in coIPing polyribosomes then leave out EDTA as it will disrupt polyribosomes.
And much much more. Any other tips from other people?
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