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Increasing protein solubility in general

protein solubility precipitate

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3 replies to this topic

#1 Michael Starr

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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



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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


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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"):



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#4 Missle



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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.

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