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His tagged protein from mammalian cells - purification of His tagged (Apr/05/2006 )

Hi all.. need some help in this one.
I am trying to purify 2 his tagged proteins of 100 and 120 KDa more or less which are supposed to be expressed in mamalian cells (Pho amp) after transfection.
I used quiagen NTA columns for purifying them but changing the lysis buffer as I wanted in their native forms. The lysis buffer that I used was RIPA buffer in the first ┬┤place , although I believe this is not convenient for maintaining the activity. I also used a buffer composed of 50 mM HEPES, 10 mM KCl, 1 mM EDTA and 1 mM EGTA plus protease inhibitor.
After a western loading control showed that some contaminants are retained in the column but after developing of the western (using anti myc as the tag has hismyc) no tagged protein was found, it even looked that was diluted with the washings..... what a situation.
My concern is how to improve the retention, and elution of the protein maintaining the activity of the protein. I would appreciate any suggestion.

Thanks in advance.


You should not use EDTA with these columns.