II am currently working on a protien to be crystallized. The protien is bound to FAD, that's why when I purify the protein with Ni affinity column its colour was yellow . but surprisingly, after changing the buffer from Tris HCL to HEPES NaOH, there was a problem with elution of prtien as SDS PAGE shows nothing.
II am currently working on a protien to be crystallized. The protien is bound to FAD, that's why when I purify the protein with Ni affinity column its colour was yellow . but surprisingly, after changing the buffer from Tris HCL to HEPES NaOH, there was a problem with elution of protien as SDS PAGE shows nothing.
Hi Sherif,
can you be a bit more specific about the buffer compositions, salt concentration, pH of the buffers used for protein binding, column washing, and column elution? There are several ways that protein can be eluted from a Nickel column if it hasn't precipitated in situ - pH change of the buffer, imidazole, EDTA etc. Also, if the protein you are working with is very hydrophobic, addition of something (detergent, alcohol) that can disrupt hydrophobic interactions to prevent non-specific interactions with the resin matrix could also be useful.
Secondly, do you know whether the protein had actually bound specifically to the column and that the yellow colour on the column wasn't endogenous bacterial proteins that also have slight colouration?
Please feel free to discuss this here further. Good luck!
the buffer conditions was
lysis buffer : 50mM tris HCl pH7.5 100mM NaCl imidazole 5mM glycerol 5%
wash buffer: 50mM tris HCl pH 7.5 100mM NaCl 5% glycerol 20mM imidazole
elute : 50mM Tris HCl 30mM NaCl 500mM Imidazole 5% glycerrol
this time everything was perfect
but when I changed tris HCl to HEPES NaOH there is nothing
O.K ; I could spend a lot of time discussing alternatives but the first thing I'm going to suggest is the simplest. Perform the purification in Tris HCl because you know it works.
Then dialyse or buffer exchange the eluted protein into your preferred Hepes-containing buffer. Hopefully this should result in the desired protein being present. I use HEPES buffers all the time for Nickel and other metal affinity chromatography without problem, so it is possibly something intrinsic to your protein of interest.
If this doesn't work, come back and we can get more creative. Good luck!
yes I will try that because this is strange for me if tris HCl only works
thank you so much
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