Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

How to do Co-IP of Triton x-100 insoluble proteins (e.g. intermediate filament p


  • Please log in to reply
1 reply to this topic

#1 victor.m

victor.m

    member

  • Active Members
  • Pip
  • 17 posts
0
Neutral

Posted 19 June 2009 - 02:27 AM

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

#2 liweixie

liweixie

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 21 June 2009 - 11:26 AM

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

Add some SDS, concentration around 0.1% to 0.5 % depends on how hard is the protein attached.....try it, SDS may help




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.