Protein refolding from Inclusion bodies - how to do and confirm the folding? (Aug/15/2006 )
Hiya!
For my protein, i use a tris buffer with sarcosine (detergent) and copper sulfate for refolding. it has a single disulphide linkage.
what other conditions do ppl here use? some papers mention arginine in refolding.. how wud that help? detergents may help solubilization i guess. anything else that is used and what does it do. plz let me know.
Also, how do u guys check for correct refolding. as of now, i have no methods for that:(
Do help:)
Thanks.
I use glycerol 10 % or glycine in my refolding buffer. sometimes people do step dialysis (slow removal of the denaturant by using succesively lower concs of it in the buffer changes but i found that my protein folded nicely in a single step dialysis, i.e i just dialysed against buffer without any urea in it.
The easiest way to check for refolding is to do an activity assay for your protein after the refolding procedure. if ti gives you activity, well great !
The easiest way to check for refolding is to do an activity assay for your protein after the refolding procedure. if ti gives you activity, well great !
Do you mean that you just do refolding in a 10% glycerol solution while dilyzing against a detergent free buffer?
if so i wil try your method! it's much more easier then usual reloding steps
about the activity test that will definetly help! but sometimes a non well folded protein can active also (case of growth factors)
I think soraya can check her samples on SDS-page : check the samples with or without b-mercaptoethanol
then if for example the protein size is around 14 KDa it will appear at 14KDa when u add b-mercapto and around 25 KDa when you don't
sometimes SDS-PAGE is not sensitive enough so westerno blotting might help better in this case
The easiest way to check for refolding is to do an activity assay for your protein after the refolding procedure. if ti gives you activity, well great !
Do you mean that you just do refolding in a 10% glycerol solution while dilyzing against a detergent free buffer?
if so i wil try your method! it's much more easier then usual reloding steps
about the activity test that will definetly help! but sometimes a non well folded protein can active also (case of growth factors)
I think soraya can check her samples on SDS-page : check the samples with or without b-mercaptoethanol
then if for example the protein size is around 14 KDa it will appear at 14KDa when u add b-mercapto and around 25 KDa when you don't
sometimes SDS-PAGE is not sensitive enough so westerno blotting might help better in this case
SDS-PAGE will only help you to differentiate between a monomer and a dimer.
if you want to see if your protein is well refolded (id est that the right intramolecular disulfide bond is formed) SDS PAGE will be useless.
I think that the activity test is the best, unless you want to refold complexes, then you can check by PAGE or by size exclusion chromatography (allowing you to purify it also)/
If your protein contain disulfide bridge and if some problems with dimers after refolding try to use GSH-GSSG ( glutation reduced\ oxidized pair) 10:1 ( 5 mM GSH ;:GSH: 0.5mM GSSG)
L arginine prevent aggregation during refolding ( one of the mechanism - binding with tyrosin residue (non covalent) so decreasing hydrophobicity of unfolded parts of the protein ( stabilization effect) If you use N-laurol sarcosine for solubilisation of protein use b-cyclodextrin ( as detergent trap) during refolding
Good luck with protein folding !