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
- - - - -

0.1-0.25% Sodium Deoxycholate vs 420mM NaCl in TrionX-100 lysis buffer. Which is

Co-IP Cell Lysis

  • Please log in to reply
4 replies to this topic

#1 asaren

asaren

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 18 May 2015 - 11:39 PM

Dear All,

 

I want lyse whole cell including nucleus of SY5Y cell without mechanical disrupting for Co-IP.

 

I have read many resources, and it can be done by either adding 0.1-0.25% Sodium Deoxycholate or increase NaCl to 420mM, in TritonX100 lysis buffer (50 mM Tris•HCl, pH 8.5, 150 mM NaCl, 1%TritonX100).

 

Which one is better to lyse whole cell for Co-IP?

 

Thank you

Cheerios,


Edited by asaren, 19 May 2015 - 12:29 AM.


#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,010 posts
439
Excellent

Posted 19 May 2015 - 01:03 AM

The answer depends on your protein. You should optimise the conditions to suit. However, in general higher salt concentrations can disrupt protein:protein interactions, as can detergents.... stay as close to physiological conditions as you can.



#3 asaren

asaren

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 19 May 2015 - 06:34 AM

The answer depends on your protein. You should optimise the conditions to suit. However, in general higher salt concentrations can disrupt protein:protein interactions, as can detergents.... stay as close to physiological conditions as you can.

 

Oh God. I'd like to lyse nuclear membrane to collect nuclear protein. Non-ionic detergent alone like Triton X-100 won't break the membrane. I know denatuirng detergent can break up some protein-protein interaction. Just know today that high salt can do too.

 

Do you homuch NaCl would consider it too high? Thank you



#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,010 posts
439
Excellent

Posted 19 May 2015 - 12:55 PM

IPs are usually done in 300 mM NaCl  or less (or equivalent osmolarity with other ionic solutions), physiological osmolarity is 150 mM NaCl, which equates to 0.9% saline or normal saline. You need to determine which concentrations you can work with for your protein.



#5 asaren

asaren

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 19 May 2015 - 09:03 PM

IPs are usually done in 300 mM NaCl  or less (or equivalent osmolarity with other ionic solutions), physiological osmolarity is 150 mM NaCl, which equates to 0.9% saline or normal saline. You need to determine which concentrations you can work with for your protein.

 

Thank you Bob1







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

©1999-2013 Protocol Online, All rights reserved.