Navigation Links
Double-Affinity Purification of GST-His Tagged Proteins Using Vivapure 8-to-96 Well Cobalt-Chelate Kit

Lucia Martinez and Ario de Marco
EMBL Protein Expression Core Facility, Meyerhofstr. 1, D-69117, Heidelberg, Germany

Introduction
Immobilized Metal ion Affinity Chromatography (IMAC) is an easy and reliable technique that allows the fast separation of His-tagged proteins from total lysates. There are several commercially available resins but all of them show some affinity for untagged proteins. Therefore, a second chromatographic step is often necessary and this implies a preventive buffer exchange step.

Affinity purification of GST-tagged proteins using glutathione sepharose usually results in lower degree of non-specific contamination, even though degradation products of the fusion proteins are often co-purified. A combined, two-step affinity purification can overcome the described drawbacks.

Here we report the purification of a 27 kDa protein fused to a double-tag (Gluthathione-S-Transferase fused to a poly-histidine tag, GST+HIS; Figure 1) using Vivapure Cobalt-loaded 8-strips. The proposed protocol does not require buffer exchange steps and includes the digestion and removal of the tags.

The first purification step exploited the GST-tag to separate the fusion protein from the bacterial proteins. The eluate was directly digested in the presence of tobacco etch virus protease (TEV protease) to remove both tags, and finally the His-tag was used to bind the double tag-moiety to the Vivapure Co-Chelate columns whereas the purified target protein was recovered in the flow-through.

Materials and methods
Cultures
A 5-ml preculture of E. coli transformed with the double-tagged fusion protein was used to inoculate 500 ml LB medium. The cells were initially incubated at 37C under agitation (180 rpm) until the OD was 0.4, then at 20C. Protein expression was induced by addition of IPTG to a final concentration of 100 M.

Pellet recovery
The cells w
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
2. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
3. Yeast Protein Production System Features High Yields and One-Step Purification
4. Expression and Purification of Recombinant Proteins That Have Native Amino Acid Sequence
5. Easy Cosmid DNA Purification with StrataPrep Kits
6. MinElute PCR Purification Kit
7. QIAquick PCR Purification Kit
8. Microarray Target Purification Kit and the Microarray Target Synthesis Kit (T7)
9. Ultra-Fast Purification of Small Nucleic Acids
10. RNAClean - RNA & cDNA in vitro Reaction Purification
11. RNAPrep - Total RNA Isolation Purification
Post Your Comments:
(Date:7/23/2014)... OncLive® is pleased to announce that ... Strategic Alliance Partnership program. , By participating ... will have the opportunity to work with the ... research initiatives, community-directed cancer prevention programs, and other ... OncLive to educate their peers by providing insights ...
(Date:7/22/2014)... (PRWEB) July 23, 2014 The ... , The goal of the Symposium is ... spectrum of scientists working in the biomaterials field, ... that represent the most promising directions for ultimate ...     Provide faculty and government scientists with ...
(Date:7/22/2014)... in water with ultrasound and they,ll spin around their ... yet knows exactly. But researchers at the National Institute ... it,s fast. At up to 150,000 revolutions per minute, ... object submerged in liquid ever reported. , The discovery ... that they could be used not only for moving ...
(Date:7/22/2014)... 22, 2014 CTD Holdings, Inc. (OTCQB:CTDH), ... medical device, cosmetics, and other markets, announced today that ... group of qualified private investors led by Novit L.P., ... , The transaction involved the signing of a Securities ... of Common Stock at a price per share of ...
Breaking Biology Technology:OncLive Expands Its Strategic Alliance Partnership Program With the Addition of UNC Lineberger Comprehensive Cancer Center 2OncLive Expands Its Strategic Alliance Partnership Program With the Addition of UNC Lineberger Comprehensive Cancer Center 3The 2014 NJ Symposium on Biomaterials Science Earns Society for Biomaterials Endorsement 2NIST shows ultrasonically propelled nanorods spin dizzyingly fast 2CTD Holdings Closes $1.725 Million Private Placement 2CTD Holdings Closes $1.725 Million Private Placement 3
... Network sat down with Tom Still, the president of ... basic tenets of the organization and initiatives Still and ... grow. Read Part One. , , ... Wisconsin Technology Council has encountered and what are your ...
... it is in chains. Fail to develop the talents of ... wealth. This quotation is taken from Tony Blairs Labour Party ... one of The Mismanagement of Talent (Oxford University Press), a ... Brown and Sara Williams. , ,The main focus of ...
... My fathers slow retreat in the face of Alzheimers Disease ... degeneration was slow, at first, but gathered momentum over time. Independence ... otherwise fine physical shape for a man of 80, but his ... word or two was gone. His body simply forgot how to ...
Cached Biology Technology:WTN exclusive interview with Tom Still, Wisconsin Technology Council - Part 2 2WTN exclusive interview with Tom Still, Wisconsin Technology Council - Part 2 3WTN exclusive interview with Tom Still, Wisconsin Technology Council - Part 2 4Talent Mismanagement 2Talent Mismanagement 3The passing of Ronald Reagan, and other Americans, is a reminder of the promise of stem-cell research 2
(Date:7/23/2014)... Honey bees, especially the young, are highly sensitive ... together to maintain temperatures within a narrow range. Recently ... at Tufts University,s School of Arts and Sciences, is ... heat within a hive in process similar to how ... vessels and skin. , "This study shows how ...
(Date:7/23/2014)... lab experiments using tissue samples cultured from cystic fibrosis ... the UNC Marsico Lung Institute have shown that a ... of another CF drug. , The finding, published today ... how a mutant CFTR protein becomes unstable and loses ... of the two drugs. The research offers several insights ...
(Date:7/23/2014)... July 23, 2014 Migratory songbirds are disappearing, and ... climate change, loss of habitat, acid rain and light ... missing: where do these birds go once they leave ... encountering along the way? , To answer this question, ... created the first ever migratory connectivity map produced for ...
Breaking Biology News(10 mins):How honey bees stay cool 2UNC researchers find unsuspected characteristics of new CF drugs, offering potential paths to more effective therapies 2UNC researchers find unsuspected characteristics of new CF drugs, offering potential paths to more effective therapies 3UNC researchers find unsuspected characteristics of new CF drugs, offering potential paths to more effective therapies 4York University researchers use bird 'backpacks' to put wood thrushes migration on the map 2
... Medicine researchers have identified biochemical changes in people taking ... These changes occur in a neurotransmitter pathway that is ... endocrine system that controls the sleep cycle, suggesting an ... The study, published on July 17, 2013, in the ...
... is to be boosted with an international ... from the University of Edinburgh and the Massachusetts-based biotechnology ... insight into the cell processes behind these debilitating conditions. ... potentially be used as treatments. The three-year collaboration ...
... are able to communicate with each other by secreting ... a critical density (the Quorum), the bacteria are able ... density has been reached certain genes are activated that ... the expression of virulence factors. Bacteria utilize this so-called ...
Cached Biology News:Biochemical mapping helps explain who will respond to antidepressants 2Biochemical mapping helps explain who will respond to antidepressants 3Transatlantic partnership to tackle neurodegenerative disease 2New methods to visualize bacterial cell-to-cell communication 2