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
- - - - -

immunoprecipitation lysis buffer


  • Please log in to reply
6 replies to this topic

#1 yoshimi79

yoshimi79

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 22 January 2010 - 08:35 AM

Does anyone know what homogenization/lysis buffer I can use to solublize the protein and disrupt protein-protein bonds (ie. I do not want to pull down complexes) but still maintain protein structure integrity and activity. I need to pull down a kinase and assay it's activity independent of other proteins.
Thanks.

#2 laurequillo

laurequillo

    The Goddamn Batman!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 265 posts
1
Neutral

Posted 26 January 2010 - 08:15 AM

You could use a ripa buffer with SDS and then wash several times using stringent conditions (high detergent concentration, high salt concentration).

I think you can do the lysis in 1% SDS and the dilute the sample to 0.1% SDS to do the Ip.

Edited by laurequillo, 26 January 2010 - 08:15 AM.

"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

"Im the goddamn batman"

#3 yoshimi79

yoshimi79

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 26 January 2010 - 11:30 AM

1% SDS won't denature the protein?

#4 madrius1

madrius1

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 114 posts
0
Neutral

Posted 27 January 2010 - 10:07 AM

Yes, it will.

I can't see a way of disrupting protein-protein bonds while keeping protein integrity and activity, though..

Kinase assays usually permit a simple IP to test protein kinas activity. Can't you use a simple IP to verify?

#5 laurequillo

laurequillo

    The Goddamn Batman!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 265 posts
1
Neutral

Posted 28 January 2010 - 11:01 AM

Sorry, I didnt think of the activity for the kinase assay...

madrius1 is right, and normally to perform kinase assays we do normal Ips
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

"Im the goddamn batman"

#6 laurequillo

laurequillo

    The Goddamn Batman!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 265 posts
1
Neutral

Posted 29 January 2010 - 03:00 AM

If you need to be sure that is indeed your protein of interest the "active" one in the kinase assay, you could check if you can produce your protein in vitro or in bacteria, and wether is as an active protein (for example the kinase I work with is active when you produce it using an in vitro translation kit), and then use it to do the kinase assay.
(This if you dont want to do a normal Ip)
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

"Im the goddamn batman"

#7 yoshimi79

yoshimi79

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 29 January 2010 - 09:10 AM

I'm trying to measure differences in activity between genetically different mouse lines, so I need it pulled out of tissue lysate.
Thanks for all your advice though.
I was just worried about pulling down other proteins that may complex with my kinase that could produce nonspecific activity in the kinase assay. The assay is an ADP glo assay which measures the level of ADP remaining after incubation of kinase+ATP+substrate. I wanted to minimize the possibility that nonspecific substrates and kinases would included in my samples.
I really appreciate all your thoughts! Thank you!




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

©1999-2013 Protocol Online, All rights reserved.