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High selectivity purification screening of histidine‐tagged proteins using small sample preparation formats containing cobalt IMAC media
Author(s) -
Björner Maria,
Lundqvist Jon,
Hedlund Helena
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.581.11
Subject(s) - cobalt , histidine , chromatography , chemistry , nickel , yield (engineering) , protein purification , metal , repeatability , selectivity , combinatorial chemistry , materials science , biochemistry , organic chemistry , enzyme , catalysis , metallurgy
The histidine‐tag is commonly used to facilitate purification of recombinant proteins. For immobilized metal‐ion affinity chromatography (IMAC) the most used metal ion is nickel. However, also cobalt has been shown useful obtaining high purity with good yield of his‐tagged proteins in IMAC applications. We have designed three formats containing cobalt IMAC media, convenient for rapid and simple small‐scale purification and screening of his‐tagged proteins. The formats, a gravity flow column, a spin column and a 96‐well plate, all allow efficient parallel purification of proteins in μg to mg scale within 40 minutes using fast and easy protocols to help ensure pure target proteins. His‐tagged green fluorescent protein expressed in E. coli was used as model protein to demonstrate the functionality of the formats. The results show that recovery and purity is > 80 % using all formats. The repeatability has proven to be high for all formats with < 10 % difference from average for each run. A comparison between cobalt and nickel IMAC media was performed and our products containing cobalt IMAC media show higher purity compared to products containing nickel IMAC media while still maintaining a high yield.