His tagged protein purification - (Nov/22/2007 )

I Am trying to purify a protein of ~100 kda with a HIs tag at N terminal. I get a lot of extra bands. I have tried various buffers, imidazole concentration, NaCl conc. but nothing is working. What should I do?
The other problem is, when i try to grow my bacteria (BL21 with pRSET) from the plate, overnight culture grows but overday culture for induction dosen't grow. However if i do overnight from glycerol stock, overday grows fine. What should I do?

Dear friend

in the matter of undesired protein (extra bands), getting read of them u cant do much. U can try cleaning the elution fraction in different methods.

1) Centrifuge filter with deffernt cut-off (e.g 50 kDa). 2) Size exclusion chromatography. 3) anti E. coli antibody-sepharose (didnt try yet)- traping E.coli

proteins.

In the matter of the growing, i am not sure- maybe your plasmid is not stable when growing from the plate. maybe u need to look for better growing media

or different concentration of antibiotics.