Navigation Links
Addition of imidazole during binding improves purity of histidine-tagged proteins

K. Hölscher, M. Richter-Roth, and B. Felden de Neumann
GPC Biotech AG, Martinsried, Germany


The purity of histidine-tagged proteins* purified by metal chelate affinity chromatography can often be improved by optimizing the imidazole concentration in the sample and binding buffer to achieve a balance between purity and yield of the protein of interest. We determined a concentration range of imidazole that minimized the nonspecific binding of untagged proteins to Ni Sepharose™ High Performance, thereby greatly improving target protein purity. We demonstrate this approach using histidine-tagged protein kinase G ([His]6-PknG) from Mycobacterium bovis.


Introduction
The (histidine)6 tag is one of the most common tags used to facilitate the purification of recombinant proteins. However, the presence of surface-exposed histidine residues or other complex-forming amino acids can lead to nonspecific binding of untagged host cell proteins to purification media. These untagged proteins elute with the target protein and must be subsequently removed. In general, the binding affinity of these contaminants is lower than that of the tagged recombinant proteins, allowing their separation from the protein of interest by using more stringent conditions.

There are several ways to reduce the binding of contaminating proteins to Ni Sepharose media. On one hand, the amount of Ni Sepharose can be adapted to the expected amount of protein to be purified. On the other hand, imidazole can be used as a competitive agent. In this article, we demonstrate the importance of imidazole as a useful tool to enhance the purity of (His)6-PknG purified with Ni Sepharose High Performance.


Determination of the optimal imidazole concentration
To determine the optimal imidazole concentration to use du
'"/>

Source:


Page: All 1 2

Related biology technology :

1. PCR Grade Deoxynucleotides Additional power to perfect your PCR experiments
2. The measurement of DNA condensation and mitochondrial changes during apoptosis and necrosis in cell based assays
3. The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System
4. The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System
5. The development of a melanocortin MC5 receptor binding assay using the LEADseeker Multimodality Imaging System
6. The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System
7. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System
8. Development of an NK2 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System
9. The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System
10. The development of a serotonin 5-HT2C receptor-binding assay using the LEADseeker Multimodality Imaging System
11. Development of an adrenergic α2a receptor binding assay using the LEADseeker Multimodality Imaging System
Post Your Comments:
(Date:1/15/2014)... January 15, 2014 The Pittcon Organizing Committee ... Food Safety Tech , an e-Journal and producer of ... agreement for the second year for the co-location of Food ... The partnership provides that the registration fee to attend the ...
(Date:1/15/2014)... RedBrick Health , a fast-growing leader in consumer ... EmblemHealth , the New York ... the RedBrick Compass health assessment, combined with the RedBrick ... members. EmblemHealth is among the first health plans in ...
(Date:1/15/2014)... 2014 The Microcompetition with Foreign DNA theory ... of these latent viruses is the Epstein Barr Virus (EBV), ... arthritis (RA) is a chronic inflammatory disease that destroys the ... that RA patients have high concentrations of EBV DNA in ...
(Date:1/15/2014)... Biotechnology Company, Limited ("TaiGen") today announced that they have ... pharmaceutical company, to develop and commercialize nemonoxacin (Taigexyn ® ... Turkey and other members of ... for the treatment of bacterial infections including those caused ...
Breaking Biology Technology:Pittcon Announces Second Year Partnership With Food Safety Tech 2Pittcon Announces Second Year Partnership With Food Safety Tech 3EmblemHealth Selects RedBrick Health to Power its Next-Generation Health Engagement and Behavior Change System 2EmblemHealth Selects RedBrick Health to Power its Next-Generation Health Engagement and Behavior Change System 3Study: Rheumatoid Arthritis (RA) Patients Have EBV; The CBCD Says this is Consistent with Microcompetition 2Study: Rheumatoid Arthritis (RA) Patients Have EBV; The CBCD Says this is Consistent with Microcompetition 3TaiGen Biotechnology Signed Exclusive License Agreement with R-Pharm for Nemonoxacin (Taigexyn(R)) 2TaiGen Biotechnology Signed Exclusive License Agreement with R-Pharm for Nemonoxacin (Taigexyn(R)) 3TaiGen Biotechnology Signed Exclusive License Agreement with R-Pharm for Nemonoxacin (Taigexyn(R)) 4
... Inc.,(OTC Bulletin Board: NGNM) announced today that it plans ... July 31, 2008., The Company has scheduled ... results on July 31, 2008, at 11:00 AM EDT. ... 689-8033 (international) at least,five minutes prior to the call. ...
... James C. Powell, former,President and Chief Operating Officer ... of Nerites Corporation., Under Powell,s guidance, SurModics ... partnerships to commercialize products.,Powell joined SurModics in 1987 ... lead the company,s transition from a diagnostics kit,manufacturer ...
... VEGAS, July 29 American Pacific,Corporation (Nasdaq: APFC ... third quarter financial results on Tuesday, August 5, 2008. ... to participate in,a teleconference to review the financial results ... held Tuesday, August 5, 2008 at 1:30 p.m.,PDT. To ...
Cached Biology Technology:NeoGenomics Schedules its Q2 2008 Earnings Release for July 31, 2008 2James C. Powell Joins Nerites Board of Directors 2American Pacific to Release Financial Results and Hold Fiscal 2008 Third Quarter Investor Teleconference 2
(Date:4/17/2014)... about eight years ago, Katia Silvera , a postdoctoral ... were on a field trip in a mountainous area in ... never seen before. , Unable to identify it, they contacted ... turned out to be an unnamed species. So Carnevali recently ... "Lophiaris" is the genus name, comprising about 40 species in ...
(Date:4/17/2014)... a novel method to help kidney stone sufferers ... treatment possible., Kidney stones represent a major medical ... left untreated, apart from being particularly painful, they ... In many patients treated successfully, stone recurrence is ... approach to diagnosis and treatment needs to be ...
(Date:4/17/2014)... expression is the process whereby the genetic information ... such as proteins, which have numerous different functions ... an important intermediary during gene expression, by relating ... mechanisms involved in manufacturing proteins. , By ... molecules present in an organism at a given ...
Breaking Biology News(10 mins):Orchid named after UC Riverside researcher 2Orchid named after UC Riverside researcher 3Rapid and accurate mRNA detection in plant tissues 2
... of £23.6M a year to the UK poultry industry but ... against the disease ?one that can be delivered while they ... vaccine virus which provides immunity to IBV has been developed ... vaccine company Intervet UK. It can be delivered to chicks ...
... simply grab carbon dioxide out of the air and covert ... plants use light as their energy source. Chemists would also ... source for their synthetic reactions, but it doesn’t work just ... Max Planck Institute for Colloids and Interfaces has now taken ...
... of Toronto is revealing biochemical processes responsible for diseases ... the way for pharmaceutical applications. , A study ... describes how U of T and Johns Hopkins University ... play an important role in human health and disease. ...
Cached Biology News:In-shell vaccine for chick disease 2Progress toward artificial photosynthesis? 2Technology reveals 'lock and key' proteins behind diseases 2
Mouse monoclonal [GB3] to Kalinin ( Abpromise for all tested applications). entrezGeneID: 3918 SwissProtID: Q13753...
Rabbit polyclonal to VEZT ( Abpromise for all tested applications). Antigen: Full length human VEZT conjugated to GST Entrez Gene ID: 55591 Swiss Protein ID: Q9HBM0...
AADAC Immunogen: AADAC (NP_001077, 201 a.a. ~ 301 a.a) partial recombinant protein with GST tag. Storage: -20 C, Avoid Freeze/Thaw Cycles *Available for distribution in the US only...
Mouse monoclonal [CIa] to Ia Antigen (Biotin) ( Abpromise for all tested applications)....
Biology Products: