Co-IP Protocols - (Apr/11/2007 )
Hi!
I'm a PhD student and have been working with a project for nearly one year now. We have found interaction partners to the protein we are interested in and wants to verify it with with co-IP. I have a few problems that I would love to get help with.
First there is a jungle of protocols of how to do co-IP, and I don't know where to begin. We have had a kit for ChIP so I have tried to get it to work for me but it doen't.
One of my biggest problems is that my protein of interest is the same size as the light chain, and even if I have two different species, doing IP with rabbit and Western Blot with mouse, I still get the heavy and light chains shown in my blots.
I don't overexpress any of my proteins, I just examine them, as they are. I crosslink the proteins in the cells with 5mM DTBP before I harvest them.
Please I really need any help that I can get, my work kind of depends on it.
I'm a PhD student and have been working with a project for nearly one year now. We have found interaction partners to the protein we are interested in and wants to verify it with with co-IP. I have a few problems that I would love to get help with.
First there is a jungle of protocols of how to do co-IP, and I don't know where to begin. We have had a kit for ChIP so I have tried to get it to work for me but it doen't.
One of my biggest problems is that my protein of interest is the same size as the light chain, and even if I have two different species, doing IP with rabbit and Western Blot with mouse, I still get the heavy and light chains shown in my blots.
I don't overexpress any of my proteins, I just examine them, as they are. I crosslink the proteins in the cells with 5mM DTBP before I harvest them.
Please I really need any help that I can get, my work kind of depends on it.
I would do 2D gel electrophoresis to separate your protein of interest from disturbing Ig subunits; as you will perform Co-IP you should be interested in characterization of Co-IP proteins f.i. by Ms/Ms;
try to get the cDNA preferably with a tag as control
You can try the TrueBlot Kit from eBioscience - it was good in my hands.
Hi,
How is your Co-IP? I hope it works great and that hope is the reason why I am writing to you. I am doing a Co-IP now. I intend to use DSP, a crosslinker, to cross link my protein of interest before cell lysis. That is exactly what you described in your question. As far as I know, most people do not use crosslinker at all. My question for you is following. Do you think whether there is any difference in terms of results between crosslink and non-crosslink protocol? Someone told me that he could not detect interaction if he did not use DSP to crosslink.
Thanks for your attention.
Jin
I'm a PhD student and have been working with a project for nearly one year now. We have found interaction partners to the protein we are interested in and wants to verify it with with co-IP. I have a few problems that I would love to get help with.
First there is a jungle of protocols of how to do co-IP, and I don't know where to begin. We have had a kit for ChIP so I have tried to get it to work for me but it doen't.
One of my biggest problems is that my protein of interest is the same size as the light chain, and even if I have two different species, doing IP with rabbit and Western Blot with mouse, I still get the heavy and light chains shown in my blots.
I don't overexpress any of my proteins, I just examine them, as they are. I crosslink the proteins in the cells with 5mM DTBP before I harvest them.
Please I really need any help that I can get, my work kind of depends on it.
One possible method to avoid seeing the heavy or light chain in an IP is using ProteinA-HRP as your secondary. This binds to the primary antibody bound to the membrane but does not identify the denatured antibody from the IP (denatured when you ran the SDS-PAGE).
To avoid seeing the light chain band, you can also use secondary Ab specifically recognize the Fc fragment only, instead of whole IgG. Jackson immunoresearch has those.