Hi all
I have a His-tagged soluble protein (8 His residues added to 90 kDa protein)
that do not bind to IMAC column based on flowthrough showing up with Western
blott. Do you have suggestions to improve the binding?
Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to
Maybe the His Tag is blocked by the folded protein.
Try using 6M Guanidine-HCL and see if it sticks.
Then you will need to find some way of refolding your protein,
if you want to crystallize it.
Theresa H. Hsu wrote:
Hi all
I have a His-tagged soluble protein (8 His residues added to 90 kDa
Your protein is either misfolded/aggregated or the his tag is chewed
off/never translated. You could try detergents etc. To improve the state of
the protein but I would also doubl check sequence for early termination
(assuming cterm) or protelysis.
Artem
On Jan 15, 2012 12:23 PM, Theresa H. Hsu
I addition to the suggestions of checking
folding/aggregation/proteolysis, you might also want to try lowering the
NaCl concentration. Whereas I like having some to prevent ion exchange
effects on the IMAC column, I have had the case of a protein that does
not bind if NaCl is present. Binds
This likely means that your imac column is acting as an ion exchanger :-)
On Jan 15, 2012 2:50 PM, Cécile Breyton cecile.brey...@ibs.fr wrote:
I addition to the suggestions of checking folding/aggregation/**proteolysis,
you might also want to try lowering the NaCl concentration. Whereas I like
Hi Theresa:
If you can make sure that your target protein is expressed. You can first use 6
M urea to denature the protein and then try to bind it to the column. If the
denatured protein can bind to the column, it seems the histag is hided inside
of the protein. It is not exposed enough to
Hi Theresa,
I am right in thinking the protein construct you are using is just 8 His
residues added to 90kDa ?
It could be (if this is the case) that the tag is not accessible for binding
to the Ni column. Sometimes you need a linker sequence between the protein for
the
His-tag to coordinate
On Sun, 15 Jan 2012 23:12:46 +, Xiaodi Yu uppsala@hotmail.com wrote:
Another thing you can try is using Cu ion instead of Ni ion. It will fasten
the binding.
Can I know what is difference in binding chemistry of Ni, Cu, Co and Fe? Is
there specific rule for binding affinity versus
Typically affinity goes down in order cu ni co zn mg
Artem
On Jan 15, 2012 7:35 PM, Theresa H. Hsu theresah...@live.com wrote:
On Sun, 15 Jan 2012 23:12:46 +, Xiaodi Yu uppsala@hotmail.com
wrote:
Another thing you can try is using Cu ion instead of Ni ion. It will
fasten the
