It depends on the particulars of the experiment. If you are trying
to solubilize a protein from inclusion bodies in a bacterial growth, then
the most common approach is to solubilize with a high concentration of ur=
ea
or guanidinium chloride and then lower the concentration of protein and
remove the denaturant. However, both which denaturant to use and how to
remove it has to be decided empirically. We routinely use a sucrose/EDTA
wash and a Triton X-100 wash before solubilizing the proteins with clean
urea/DTT, but we always check by SDS-PAGE that the protein is in the urea
wash, not the other fractions.
This general approach often works with small, monomeric proteins.
There is an ACS publication, # 470 edited by G. Georgiou and E.
Bernardez-Clark, which has alot of information, as does an article by R. =
C.
Thompson in Meth. in Enzymol., Vol. 185. I see no reason why this approa=
ch
might not work on pure proteins in other situations.
I realize that I posted a similar reply recently, so I hope no one
feels bored or cheated.
Good luck.
Chris Halkides
>Maria:
>
> There are no simple answers as it depends on the purity of your protein
>and the characteristics of your protein. I am sure others will give you=
a
>good list of things to try. However, of the many approaches, I find the =
use
>of 2M urea in a buffer the most effective and reversible.
>
>Hope this helps!
>
>
>Tony
>
>
>At 07:27 PM 6/23/99 +0200, Maria Lorenzi wrote:
>>Hi, I am Maria Lorenzi.
>>I would like to know if there are particular methods to solubilize a pr=
otein
>>without loosing its activity.
>>Thanks
>>Maria
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>"Soltanto una cosa =E8 veramente importante da fare; ho cercato ma
>>non sono riuscita a fartela vedere. So comunque che un giorno la vedrai
>>perch=E9, come il sole, =E8 per tutti"
>>
>> M.L.
>>***********************************************************************=
**
>>Dott.ssa Maria Lorenzi
>>Dottoranda in Biochimica e Biofisica
>>Istituto di Biochimica
>>Facolt=E0 di Medicina e Chirurgia
>>Facolt=E0 di Agraria
>>Universit=E0 degli Studi di Ancona
>>Via Ranieri 60131 Ancona, Italia
>> Tel(lab):+39 71 220 4395
>> +39 71 220 4681
>> +39 71 220 4677
>> Fax: +39 71 280 2117
>> e-mail: mlorenzi@ascu.unian.=
it
>>***********************************************************************=
***
>>
>
>
>************************************
>Dr. Anthony T. Yeung, Ph.D.
>Director, Fannie E. Rippel Biotechnology Facility
>Member, Institute for Cancer Research
>Fox Chase Cancer Center
>7701 Burholme Ave. Philadelphia, PA 19111
>Voice: 215-728-2488
>FAX: 215-728-3647
>email: AT_Yeung@FCCC.edu
>http://www.fccc.edu/research/labs/yeung/
>
>*****************************************************
>This electronic mail transmission contains confidential
>information intended only for the person(s) named.
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>by another person is strictly prohibited.
>*****************************************************
Christopher Halkides
Dept. of Chemistry, UNCW
601 S. College Road
Wilmington, NC 28403-3297
(910) 962-7427