for protein refolding - (Sep/19/2007 )

Hi to all,
I have solubilized my protein which will be expressed in inclusion bodies by using Urea and Gn HCl method. In both cases bands in SDS are same intensity but probelem in refolding. If I want to go for refolding which method and which solubilized protein(Urea or Gn HCl) gives the more yeaild.

Plese send ur suggestions as soon as posible.

Thank you very much.

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First of all It depends on your protein. So refolding procedure should be optimized to your protein. General approaches you may find here refold database . also try to search your protein in this database ( it is possible you will find refolding procedure for your protein) . Here is a lot of interesting refolding procedures.

It is start up options

Good luck!

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hi

Thank u very much I will go through that and i will contact u


Thank u

bye

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in addition to circlepoint, if you do not find your protein of interest, you have to evaluate several protocols, 96-well kits are available form f.i. novobiochem/merck;

follow discussions on this site

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Hi

I could get my protein in soluble form in Urea and Gn HCl method but question is that I fi wan to purifi the large quntity of protein Which method (Urea or Gn HCl) and which method is useful to get high yeild of biological active (properly refolded) protein?


Send the sugestion as soon as posible.


Thank you, bye.

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Sorry to be unhelpful, but that question falls into the 'How long is a piece of string' category. There really is not way of telling what will happen without trying the experiment yourself (after all, that's why we call ourselves researchers and experimentalists). Thankfully, inclusion bodies are quite protective of the contents, so you can store them at 4C for a day while you work out your next step.

Here's what I'd do. Resuspend your inclusion bodies, and take off a small volume. Split this in two and treat each half with either urea or GnCl. Try your resuspension protocol, and run a gel, and maybe a functional assay if you have it. Then apply what you have learned to the bulk of the inclusion body prep.

How are you planning on diluting out the denaturant?

Oh, one more thing: Good luck! It might not work at all, but then again, it might work brilliantly!!!!!

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