How to do Co-IP of Triton x-100 insoluble proteins (e.g. intermediate filament p - (Jun/19/2009 )
Dear All,
I would have a technical question regarding co-/immunoprecipitation of proteins contained in Triton x-100 (TX-100) insoluble fraction (proteins that is very difficult to dissolve).
A protein (an enzyme) of my interest is always in TX-100 insoluble fraction which contains for example intermediate filaments (e.g. cytokeratins) and other debris. That means, that my protein interacts with some cytoskeletal proteins. I would like to do co-immunoprecipitation of my protein to get to know with what it is interacting or immunoprecipitation of e.g. cytokeratins to see whether my protein interacts with them.
My question:
How to do Co-IP of cytoskeletal proteins that it is very difficult to dissolve in whatever IP buffer? These are really debris that are normally in triton x-100 or NP-40 insoluble, so I do not know what to do (how to dissolve), if I want to incubate these proteins with antibody, A/G-agarose beads etc. to do immunoprecipitation.
Do you have any experience?
Thanks in advance.
Best regards,
victor
victor.m on Jun 19 2009, 02:27 AM said:
I would have a technical question regarding co-/immunoprecipitation of proteins contained in Triton x-100 (TX-100) insoluble fraction (proteins that is very difficult to dissolve).
A protein (an enzyme) of my interest is always in TX-100 insoluble fraction which contains for example intermediate filaments (e.g. cytokeratins) and other debris. That means, that my protein interacts with some cytoskeletal proteins. I would like to do co-immunoprecipitation of my protein to get to know with what it is interacting or immunoprecipitation of e.g. cytokeratins to see whether my protein interacts with them.
My question:
How to do Co-IP of cytoskeletal proteins that it is very difficult to dissolve in whatever IP buffer? These are really debris that are normally in triton x-100 or NP-40 insoluble, so I do not know what to do (how to dissolve), if I want to incubate these proteins with antibody, A/G-agarose beads etc. to do immunoprecipitation.
Do you have any experience?
Thanks in advance.
Best regards,
victor
Add some SDS, concentration around 0.1% to 0.5 % depends on how hard is the protein attached.....try it, SDS may help
