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:1/23/2015)... 23, 2015 Biofertilizers stimulate microbial activities ... nitrogen and phosphorous through the activities in the soil ... increasing due to the stress on reducing environmental pollution ... The report defines and segments the bio fertilizers market ...
(Date:1/22/2015)... 22, 2015  Transwestern | RBJ today announces the firm brokered ... for Shire a leading biopharmaceutical company, at Two Ledgemont Center ... Robert Richards , president, and Brian Cohen , ... entire five-floor building at 95 Hayden Ave. Photo ...
(Date:1/22/2015)... Protocol Networks brings independent technology consulting to the ... consultants, Protocol Networks has recently announced its expansion into ... his company has attracted several clients in The Constitution ... as others. With the success of these engagements, Rogers ...
(Date:1/22/2015)... 22, 2015 Controlled Substance Compliance Services ... helps companies check the legal requirements around using controlled ... China. , As their international operations expand and ... software solutions built as a result of the first ...
Breaking Biology Technology:Biofertilizers Market Expected to Reach $1,649.7 Million by 2019 - New Report By MarketsandMarkets 2Biofertilizers Market Expected to Reach $1,649.7 Million by 2019 - New Report By MarketsandMarkets 3Biofertilizers Market Expected to Reach $1,649.7 Million by 2019 - New Report By MarketsandMarkets 4Transwestern | RBJ Advises Shire in 202,000 SF Lease, Creating Boston's Largest Suburban Biotech Campus 2Protocol Networks Brings Independent Technology Consulting to the Constitution State 2Global Compliance Service for Controlled Substances to Expand to China 2
... Biotechnology Company Adopts Pharsight,s Groundbreaking ... Automation Software, ... a leading provider of software and strategic,services for optimizing clinical drug ... WinNonlin AutoPilot.,This sale marks Pharsight,s third new customer win since the ...
... /PRNewswire-FirstCall/ - GeneNews Limited (TSX: GEN),a company ... for the early detection of diseases and ... started collecting patient samples,for clinical validation studies ... In June 2006, GeneNews presented training set ...
... 17 /PRNewswire-FirstCall/ - Helix BioPharma Corp.,(TSX, FSE: "HBP") ... purchase, by way of private placement, of a ... for gross proceeds totaling CDN$16,867,200.,Proceeds from the placement ... the Company,s research and development initiatives., The ...
Cached Biology Technology:Pharsight Signs Third WinNonlin(R) AutoPilot(TM) Customer 2Pharsight Signs Third WinNonlin(R) AutoPilot(TM) Customer 3GeneNews initiates patient sample accrual for validation studies of colon cancer blood test 2GeneNews initiates patient sample accrual for validation studies of colon cancer blood test 3Helix BioPharma announces $16.9 million private placement of common shares 2
(Date:1/22/2015)... Research and Markets ( http://www.researchandmarkets.com/research/wxdglm/global_wearable ) ... Wearable Technologies Market and Applications, Opportunities, Industry Analysis, ... 2014-2020" report to their offering. , ... can be worn on the user,s body and ...
(Date:12/22/2014)... SHELTON, Conn. , Dec. 22, 2014  NXT-ID, Inc. (NASDAQ: ... a biometric authentication company focused on the growing mobile commerce ... consultant, Apollo Robbins for the 2015 International CES ... Apollo Robbins will be at the NXT-ID booth ...
(Date:12/19/2014)... and Markets ( http://www.researchandmarkets.com/research/86ncd6/micro_market ) has announced the ... Security Systems Market" report to their offering. ... perimeter security market is estimated to grow at a CAGR ... holds a larger share in this market, Canada ...
Breaking Biology News(10 mins):Global Wearable Technologies Market and Applications, Opportunities, Industry Analysis, Segmentation, Size, Growth, Share, Overview, Trends and Forecast 2014-2020 2NXT-ID Hires Master Pickpocket and Security Consultant Apollo Robbins for Wocket Smart Wallet CES 2015 Debut 2NXT-ID Hires Master Pickpocket and Security Consultant Apollo Robbins for Wocket Smart Wallet CES 2015 Debut 3NXT-ID Hires Master Pickpocket and Security Consultant Apollo Robbins for Wocket Smart Wallet CES 2015 Debut 4Micro Market Monitor: North America Perimeter Security Systems Market (Intrusion Detection Sensor, Video Surveillance, Communication/Alarm and Notification, Access Control System) 2Micro Market Monitor: North America Perimeter Security Systems Market (Intrusion Detection Sensor, Video Surveillance, Communication/Alarm and Notification, Access Control System) 3
... And, at least for mice, it's one that is best made ... designed help scientists understand the brain chemicals that guide mate selection, ... a male mouse alone or a male mouse with a female. ... females. , "Our data suggest that female mice may use, or ...
... Infectious Diseases (NIAID), part of the National Institutes ... a large international HIV/AIDS trial comparing continuous antiretroviral ... of CD4+ cells has been stopped. Enrollment was ... twice the risk of disease progression (the development ...
... Texas M. D. Anderson Cancer Center have created a ... to find, highlight, and deliver genes to tumors in ... journal Cell, is potentially an important step in making ... it may also allow prediction and monitoring of how ...
Cached Biology News:When mice choose mates, experience counts 2International HIV/AIDS trial finds continuous antiretroviral therapy superior to episodic therapy 2International HIV/AIDS trial finds continuous antiretroviral therapy superior to episodic therapy 3International HIV/AIDS trial finds continuous antiretroviral therapy superior to episodic therapy 4New hybrid virus provides targeted molecular imaging of cancer 2
Agarose, pulse field, 100 g. Nuclease-free.Gel point (1.5%): 36 1.5 C, EEO (-mr): 1800 g/cm2. Category: Nucleotides & Enzymes & Biochemicals, Ultrapure Biochemicals ....
Molecular Biological Resources has extensive abilities cloning and expression of prokaryotic and eukaryotic proteins. Please contact lloconne@molbiores.com to discuss your project. Contact lloconne@...
Bethyl Laboratories packages antibodies, conjugates and calibrators to provide quantitative ELISA kits. Each kit contains the following components, sufficient for 1000 single well assays....
quantitation of phosphatase activity, evaluation of hits and leads from HTS...
Biology Products: