how to refold insoluble protein after purification - (Feb/12/2008 )

Hi.

I am working on an insoluble viral glycoprotein expressed in e. coli. It forms inclusion bodies.I need to purify my protein according to the denatured protocol and I intend it for raising polyclonal antobodies in rabbit. How do I know how I can refold my protein effectively and how can i check that its properly folded?

Many thanks.

elsh

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Hi

i am working with protein that forms inclusion bodies for the same purpose as u.

i just dialyzed my protein against PBSX1 and send for injection. i dont have any possibility to

verify the protein refolding or activity, so it is just like a gambling (but its not my money so what i care)

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Try this site. http://refold.med.monash.edu.au/

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I am just worried about whether it will refold back according to its native structure. There is no way I can check because my protein is not an enzyme, where I can at least check its activity to ensure efficiency.

I have also read that it is usually not necessary to refold back proteins that need to be used for Ab raising. Is that true?

I also have another problem. I have raised antiserum to my virus in mice but am not getting any results from my Western blot when I used the sera as the primary Ab.I used a dilution of 3:2000. Is it too diluted?

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Have you tried to optimise the expression, so your protein doesn't go into IBs? Codon bias can force partially-synthesised proteins to go there, as can too high an expression temperature. Try dropping the temperature, using less IPTG, or if you have a codon bias problem, transform into a strain like Rosetta BL21.

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Hi swanny,

thanks for your reply. I have tried optimising by reducing temperature, rpm and iptg concentration and have gotten protein in the soluble fraction. Problem is, its only in trace amounts so there is no way i can use it except if i purify in a fermentor. the ratio for amount of insoluble to in soluble is about 1:20

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Hi amtash,

I would like to know if your raised antiserum binds to your native protein when you do Western blotting?

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hi elsh,
its vijay here!!
i am new in this field and got only 2 yrs experience over all. But i have worked on insoluble proteins and read aboout it. I dont think protein has to refold to its native form for it to get recognised by its antibody. people have used denatured protein cut and extracted from the SDS-PAGE for antibody production.
There are various protocols available for protein refolding.
Check out www.refold.com

cheers,
vj5

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Hi vijay,

I want to raise polyclonal Ab with the purpose of the serum recognizing my native protein. so if I raise it using a denatured protein can the antiserum recognise my native one?

Thanks so much for your reply

-erin

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Hi

sorry for the late answer

yes is the answer. antiserum from rabbits had beenrecognized and bind my native protein perfectly.

its also acceptable to use denaturated proteins injected into rabbits.

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