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
- - - - -

Purification using Low-Salt Lysis Buffer & Downstream app.


  • Please log in to reply
2 replies to this topic

#1 dcch

dcch

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 32 posts
1
Neutral

Posted 21 August 2010 - 09:23 AM

Hi. I discovered that my GST-fusion protein can only be present in soluble fraction at NaCl concentration as low as 30mM of lysis buffer. If I would like to purify my protein using GST binding resin, will such a low-salt condition bring disadvantage to my purification? Since it only solubilize at low-salt condition, I think I only can use low-salt in washing and thrombin cleavage buffer for subsequent purification step, because it will aggregate once present in high-salt condition, right?

Usually, the salt in lysis buffer refers to NaCl. Can I substitute it with KCl, MgCl2? Or they all will just have the same effect?

The protein will be subjected to enzymatic assay. The enzymatic assay buffer contains moderate salt (~100mM). Do I have to reoptimize the enzymatic assay buffer? I am worried if the protein will instantly aggregate once I add into the assay reaction buffer!

All opinions are welcomed! Thanks!

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,758 posts
130
Excellent

Posted 22 August 2010 - 09:41 PM

substituting kcl or mgcl2 will cause their own problems. kcl is generally more potent than nacl in its effect on proteins, mgcl2 has other effects. stick with nacl.

lower concentrations may effect non-specific binding to the resin but may still be okay (try it).

lowering salt in the assay should have little to no effect, depending on the enzyme (some may change conformation). try it at varying salt concentrations.
talent does what it can
genius does what it must
i do what i get paid to do

#3 dcch

dcch

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 32 posts
1
Neutral

Posted 04 September 2010 - 06:40 PM

substituting kcl or mgcl2 will cause their own problems. kcl is generally more potent than nacl in its effect on proteins, mgcl2 has other effects. stick with nacl.

lower concentrations may effect non-specific binding to the resin but may still be okay (try it).

lowering salt in the assay should have little to no effect, depending on the enzyme (some may change conformation). try it at varying salt concentrations.

Thank you mdfenko for the reply :)
Since my protein become more soluble when I reduced the salt concentration in lysis buffer, does it mean my protein not being expressed as inclusion bodies in E.coli? Is there any methods to know whether the protein is actually being expressed in soluble form, inclusion bodies, or the insolubility(aggregation) is due to lysis buffer?

My lysis buffer consists of 50mM Tris-HCl (pH8), 30mM NaCl, 10% glycerol and BME. Is there any parameters I can play with? Or any chemical/additives can be added to help this condition?

Lastly, even though more soluble protein was obtained, I failed to bind it to GST binding resin. I really have no idea what to do further. I have considered to refold the protein. But our lab has never do protein refolding. We are lacking of experience, protocol and even equipment. :(




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

©1999-2013 Protocol Online, All rights reserved.