Blocking antibody - (Nov/05/2008 )
I am confused about the types of antibodies. I need to do an experiment to test which receptors/ligands are involved in cell-cell interaction. Can I use antibodies designed for western blot or flow cytometry as blocking antibodies? How can I test whether the receptors/ligands are completely blocked without triggering any unwanted signalings? Thanks in advance for answering.
Tough question to answer without knowing the receptors and ligands you are interested in.
Blocking antibodies are designed for just that purpose, to block without triggering signaling. Most antibodies optimized for flow cytometry DO NOT accomplish this, as demonstrated in various competition assays. In fact some of these flow Abs will actually cross-link receptors and induce signal transduction, potentially activating your cell. I'm not an expert in Western blot Abs, but it seems to me that these Abs have been designed to recognize denatured proteins, and therefore will probably not recognize the intact protein that will be in your assays.
Bottom line, if you need a blocking antibody you should try and search the literature.
One option for you would be to try to get your hands on soluble ligand and look for changes in your cell with varying concentrations. The other way you can look to see if a receptor - ligand is important in your system is to get these cells from knockout mice.
Hope that helps!