Protein purification from Inclusion Bodies

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Pinal Pandya
Pinal Pandya's picture
Protein purification from Inclusion Bodies

Hi there,
I am trying to solubilize my protein from Inclusion bodies. I used DH5 alpha strain for the expression. And i found this protocol online for protein refolding. It says i need to dialyse the protein using urea. But my problem is i dont know where to buy the dialysis bags from. I am stumped !!! Can anyone help me.
Thanks
Pinal

R Bishop
R Bishop's picture
Pinal,

Pinal,

I find that Pierce has some nice dialysis products. I use the Snakeskin for large amounts of solution and the slide-a-lyzer cassettes for smaller volumes.

http://www.piercenet.com/products/browse.cfm?fldID=040101

Also trying using the Solution Search link on this page as there are many threads about inclusion bodies and refolding

Hope that helps

Rb

Pinal Pandya
Pinal Pandya's picture
Thank you soo much for your

Thank you soo much for your help RB , this definitely helps a lot and i am going to check out the solutions search link.

thanks again

Pinal Pandya
Pinal Pandya's picture
Thanks for helping me before

Thanks for helping me before RB. I have another question, I found this protocol online for solubilization and refolding. the method is as follows: add 100mM tris buffer ph 8, 50mM glycine ; sonicate; dissolve supernatant in the above buffer + 8M urea.

then add 5mM reduced glutathione and 0.5 mM oxidized glutathione. and then proceed for dialysis.

so i guess my question is why do we need ot add glutathione at all? reduced and oxidized? and if we use oxidized glutathione with ~6% ethanol , is it ok?

Please help me!!!
Pinal

Pinal Pandya
Pinal Pandya's picture
hi again

hi again

i was wondering if anyone here has used the inclusion bodies purification and renaturation kits:

Pro-Matrix Protein Refolding Kit (PIERCE)
Rapid GST inclusion body solubilization and renaturation kit (cell Biolabs Inc)

if anyone has used any of these before, could you tell me if it worked?

i want to know if these are worth investing any money in.

thanks
pinal

TigerShark
TigerShark's picture
I have had many difficulties

I have had many difficulties with these kits. Many of them are based upon diluting denatured inclusion bodies into "intermediate" folding buffers as part of a screen. These buffers contain less denaturant and usually additives like arginine, sucrose, or cyclodextrin etc... Many of these conditions work fine for retaining solubility, but the problem is that you're still stuck with having to get your protein into the buffer that you want, usually something like Tris or PBS. At that point, with my protein, everything fell apart, the protein precipitates right out of solution etc..... so you're back to square 1 again.

In my experience, dilution to a very low concentration and constant filtration of beginning aggregates has been the most helpful solution yet.

sriram
sriram's picture
hi .......

hi .......
iam having problem with solubilisation of protein from inclussion bodies with 0.2%sds in 50 mM tris with ph 7.5 could u tel me how much buffer should i use to get compleate solublise the protein to each gram of protein

sriram
sriram's picture
 

 

davedilyx
davedilyx's picture
Solubilization often requires

Solubilization often requires you to screen different pH, salt, organic solvent, polymer, reducing agent etc. if possible in a combination. Check out these protein solubility screening kits for a selection of protein solubilizaiton kits.
Cheers,
Dave

qiang wang
qiang wang's picture
I usually solubilize my IBs

I usually solubilize my IBs with my buffer + 6M urea, and the target proteins are usually easily purified and refolded by dialysis.