Protein Purification of an His-Tag Protein using a Ni Column - (Jan/12/2011 )
Hi guys,
I was performing the first chromatography on my protein purification tonight and I accidentally forgot to add the 40 mM imidazole and 500 mM NaCl. After having the system running, I realized that I forgot to add that and I decided to collect the sample that went through the column. That sample was then remixed with my original sample and I added then the imidazole and NaCl. Do you think that is a problem for my experiment or will it work just fine since that I didn't waste anything.
Please let me know since that I am worried that I could have ruined the entire experiment.
Thanks in advance.
-biomedica-
biomedica on Thu Jan 13 01:44:20 2011 said:
Hi guys,
I was performing the first chromatography on my protein purification tonight and I accidentally forgot to add the 40 mM imidazole and 500 mM NaCl. After having the system running, I realized that I forgot to add that and I decided to collect the sample that went through the column. That sample was then remixed with my original sample and I added then the imidazole and NaCl. Do you think that is a problem for my experiment or will it work just fine since that I didn't waste anything.
Please let me know since that I am worried that I could have ruined the entire experiment.
Thanks in advance.
Hola, I think you wouldn´t have a big problem if the concentration of your protein in high.Imidazole at low concentration is to avoid bounding of inespecific His (less than six) and high salt prevents inespecific adsortions. To overpass this mistake you have to extensivelly wash the column makins sure that baseline is recovered, after that elute, analyze and have a bit of luck.
-protolder-