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-   -   I need a solution for purification of an his-tagged protein (http://www.molecularstation.com/forum/protocols-methods-forum/18442-i-need-solution-purification-his-tagged-protein.html)

Bazeille Nicolas 04-25-2006 04:39 PM

I need a solution for purification of an his-tagged protein
 
Hello everybody,


For a long time I try to purify a his tagged protein. At the beginning, I load
the cells extract in a Nickel column. The result are great, about 10 mg of
protein per liter of culture. Unfortunatly, it still rest a small amount of
contaminants.

I 've tried to load the rich-protein fraction in an heparin column but it does
not work well, no more contaminants are eliminated. So, I check for others
solution wich are :

immunoaffinity column : Affi-gel (Biorad)
Gel filtration : Sephadex G-75
gradient of ammonium sulfate precipitation according to Englard et al.

However, I'll take so much time...

So, maybe, someone possess some others easier solutions to help me in
purification.
Thanks a lot.


Wolfgang Schechinger 04-25-2006 07:55 PM

I need a solution for purification of an his-tagged protein
 

Dear Bazeille,

How did you elute your protein from the Ni column? Mean, did you elute
with increasing concentrations of imidazole to get as much unspecific /
unwanted stuff off as possible? Then, elute it either with further
increasing imidazole oder just EDTA (then you have to re-charge the
column with eg Nickel chloride or -sulfate)

My suggestion for the workup is a stepwise ammomium sulfate (AS)
precipitation (actually, I had done it before the Ni column as you may
remove a lot of unwanted and interfering stuff without much effort).
You may start with 30% saturation and increase the AS in 5% steps up to
70%. You may add solid AS in order to keep the volume small, but add it
slowly.
You might optimize the procedure with an aliquot if your protein and
analyze the fractions by SDS-PAGE after dialysis and then run the big
prep in batch mode.

Gel filtration is a good or bad idea, depending on the size of your
contaminant(s).
Ion exchange on a mono-Q or DEAE-agarose and elution with a salt (eg
NaCl) or pH gradient might work well then for further cleanup. For
'polishing' then HIC or another affinity step, depending on the
properties of your protein.

Best,

Wo


Bean Long 04-25-2006 11:14 PM

I need a solution for purification of an his-tagged protein
 
Bezeille,

Did you use some imidazole in your Ni column binding buffer?? 5-20 mM
imidazole in the binding buffer should help eliminate some of the
non-specific binding.

--
Bean

Remove "yourfinger" before replying

Lisa 05-03-2006 11:09 PM

I need a solution for purification of an his-tagged protein
 
Have you tried Dynabeads from Dynal? Er.. Invitrogen now.

They're coated magnetic beads that will specifically bind His-tagged
proteins. If you go to the American Invitrogen website, they're listed
under sku # 101-01D or 101-02D

Hope it helps,

Lisa

Bazeille Nicolas wrote:



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