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
- - - - -

Increasing protein solubility in general

protein solubility precipitate

  • Please log in to reply
3 replies to this topic

#1 Michael Starr

Michael Starr

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 59 posts
1
Neutral

Posted 15 April 2016 - 01:50 PM

I am looking for general techniques/additives to buffer to increase protein solubility and reduce precipitation. Our lab has had on and off issues with protein precipitation, and I figured if we could combat that that would increase our yields.

 

I know there are techniques for increasing solubility of transgenic proteins in E coli, such as co-expression of chaperonins, but I am looking for more in vitro, for any given protein.

 

So far I have that L-Arg and L-Glu can help at 50 mM.

What about Tween 20? Would that do anything?

 

Other buffers to try? pH and salt concentration would probably be protein specific, yeah?

 

I remember seeing a bunch of listed suggestions online somewhere but I can't find it again.



#2 labtastic

labtastic

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 205 posts
22
Excellent

Posted 17 April 2016 - 11:15 AM

Some things to think about....

 

Trying working with your protein as a fusion with another more soluble protein (e.g. GFP, SUMO, GST etc)

 

Try a different construct of your protein if you are expressing it recombinantly. Sometimes truncating residues at either end can dramatically change protein stability.

 

Try a different ortholog of your protein. Sometimes the same protein from two different organisms can have markedly different stability.

 

Sometimes higher salt concentrations can help keep proteins from interacting too much with themselves (causing precipitat)e. Not sure what you are currently using, but consider using up 500mM NaCl. Or try KCl.

 

Adding glycerol 5-10% can help

 

Small amount of tween or triton (0.1%) can help but best to stay away from detergents if you can

 

Different pH's and buffers (Tris, HEPES, MOPS, MES, phosphate, Bis-Tris, Tricine, etc)

 

Does protein have co-factors? Calcium, magnesium, metals, nucleotides, FAD, NAD? 



#3 mdfenko

mdfenko

    an elder emeritus

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,383 posts
220
Excellent

Posted 18 April 2016 - 06:25 AM

it's very difficult to generalize solubility of various proteins. some are more soluble at high salt, some less. go to high with salt and you'll precipitate the protein similarly to the use of ammonium sulfate.

 

the pI of proteins vary but most are in the range of pH 4-6. so you can hold most proteins at physiological pH.

 

various additives can be tried, such as ethylene glycol,

 

if you don't mind denaturing the protein then you can use ionic detergents like sds or chaotropic agents such as urea and guanidinium chloride.

 

there are some handbooks available for download from ge lifesciences that may give you some guidance (eg "purifying challenging proteins"):

 

http://www.gelifesci...port/handbooks/


talent does what it can
genius does what it must
i used to do what i got paid to do


#4 Missle

Missle

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 166 posts
27
Excellent

Posted 19 April 2016 - 05:43 AM

I agree with what was said above.  Additionally, the single most impactful factor (in my opinion) is temperature.  Expressing the protein at 18C - 25C for longer periods of time will improve solubility of many proteins.







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

©1999-2013 Protocol Online, All rights reserved.