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A Novel Rapid Procedure for Purification of IgY from Egg Yolk

There are several advantages with the production of avian antibodies. The immunisation of chickens induces the production of specific antibodies in both serum and egg yolk in similar concentrations. ...

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

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 ...

Automated microfiltration using AKTAcrossflow

In this application, we outline steps for optimizing the purification of: 1) ß-glucosidase from Pichia pastoris cell culture, a relatively simple application working from whole cells, and 2) ...

Capto S Cation Exchanger for post-Protein A purification of monoclonal antibodies

This application note demonstrates the use of Capto S in a MAb process as a second step after Protein A affinity chromatography using MabSelect SuRe. Capto S gives high dynamic binding capacity for ...

Desalting and buffer exchange with Sephadex G-25

Gel filtration has successfully been employed for size based separations of macromolecules since the late 1950s. Gel filtration, or size exclusion chromatography, may be performed in three ...

Efficient isolation of protein fragments for structure analysis; ÄKTApurifier

A novel, high pressure, high performance chromatography system, ÄKTA ™ purifier and UNICORN ® , was used for efficient isolation of protein fragments for structure analysis. The pre-programmed ...

Efficient, rapid protein purification and on-column cleavage using GSTrap FF columns

A purification strategy involving the production of soluble, purified proteins in a single step would be an invaluableresource and indispensable in many laboratory environments. This Application Note ...

Proteomic analysis of the human plasma proteome using Ettan MDLC

Ettan MDLC is a high-resolution chromatography system with a wide dynamic range to enable analysis of complex mixtures containing both high- and low-abundance proteins and is designed for peptide ...

Fast and simple purification of histidine-tagged proteins using His GraviTrap

His GraviTrap gives the following performance benefits: Five-times faster purification than the comparable product from another supplier Possibility to purify both clarified and unclarified ...

Hemoglobin A1c Measurement with Mono S Method

Mono S™ 5/50 GL (Tricorn™) columns are suitable for the measurement of HbA1c and the calibration of healthcare instruments that analyze HbA1c. The Mono S method is the Swedish designated method since ...

High-productivity capture of alpha-chymotrypsin on Capto S cation exchanger

This application note describes the capture of a-chymotrypsin from clarified homogenate of Escherichia coli using packed bed chromatography with the recently developed strong cation exchanger Capto™ ...

High-productivity Capture of Green Fluorescent Protein on Capto Q

This application note describes the capture of Green Fluorescent Protein (GFP), recovered from homogenized and clarified Escherichia coli, using packed bed chromatography on the newly developed ...

Highly sensitive phosphopeptide analysis using Ettan MDLC and a linear ion trap mass spectrometer

Ettan™ MDLC coupled to a linear ion trap mass spectrometer was used to characterize phosphopeptides in the low femtomole range. Phosphopeptides in a complex biological sample were sequenced, and the ...

Highly specific and rapid immuno-precipitation of GroEL protein and PDGF b receptor from cell lysates using Immunoprecipitation Starter Pack

Immunoprecipitation Starter Pack, which contains 2 ml each of Protein A Sepharose ™ 4 Fast Flow and Protein G Sepharose Fast Flow media, provides the means for convenient, efficient, and highly ...

HisTrap FF crude for faster purifcation of histidine-tagged proteins

Features of HisTrap™ FF crude include: No need for centrifugation or filtration of the samples. Direct sample application. No difference in final purity and recovery when using: – clarified or ...

Improved Purification of Histidine-tagged Proteins with Ni Sepharose High Performance

The (histidine)6-tag has become the most used affinity tag due to its small size, strong metal ion binding, and ability to bind under denaturing conditions. Immobilized Metal Ion Affinity ...

Increased dynamic range in quantitative proteomics using CyDye labelling, Ettan LC for 2D micropreparative chromatographic separation, and SDS-PAGE.

This study demonstrated a proof of principle for a novel 2D LC based approach for quantitative proteome analysis, simultaneously detecting and quantitating abundance ratios of stable isotope labelled ...

Integrating UNICORN and DeltaV

Integrating UNICORN™ from GE Healthcare and DeltaV™ platform from Emerson; a leading pharmaceutical company has developed a new full GMP controlled facility for the manufacturing of purified ...

MabSelect SuRe studies on ligand toxicity, leakage, removal of leached ligand, and sanitization

MabSelect SuRe™ is an affinity medium designed for purification of large volumes of monoclonal antibodies. The alkali-stabilized protein A-derived ligand allows extensive use of sodium hydroxide for ...

Method optimization and scale-up of the purification of recombinant bovine carbonic anhydrase with IMAC Sepharose 6 Fast Flow

This study demonstrates that IMAC Sepharose™ 6 Fast Flow is well adapted for laboratory- to process-scale purification of untagged proteins with good selectivity and recovery of the target protein. ...

One-step purification of monoclonal IgM from cell culture supernatant

The development of a general purification protocol for immunoglobulin M (IgM) has met with problems, as the unique variation in the amino acid sequence of the variable regions give different ...

Optimization of loading conditions on Capto adhere using design of experiments - Process-scale antibody purification

Capto™ adhere is a strong ion exchanger with multimodal functionality designed for intermediate purification and polishing of monoclonal antibodies. Removal of remaining contaminants is achieved in ...

Optimization of the purification of mono-PEG40 avidin with MacroCap SP

This application note describes the purification of mono-PEG40 avidin (mono-poly[ethylene] glycol 40000 avidin) using MacroCap™ SP cation exchange medium. MacroCap SP is designed to separate large ...

Optimizing elution conditions on Capto MMC using Design of Experiments

The multimodal ligand of Capto™ MMC generally requires elution conditions that differ from those used with traditional ion exchangers. This application note describes how Design of Experiments (DoE) ...

Optimizing Protocols for Automated Multistep Purification of (histidine)6-tagged Protein using ÄKTAxpress

ÄKTAxpress™ is a chromatography system for automated multistep purification of large numbers of (histidine)6- and Glutathione S-transferase (GST)-tagged proteins. Pre-optimized methods are easily ...

Partial purification of DNA binding proteins using HiTrap Heparin

This work describes partial purification of three different DNA binding proteins, i.e. H-NS, RNA polymerase and Oct-1, using pre-packed HiTrap Heparin 5 ml columns in the initial chromatographic ...

Peptide Mapping at Low and High pH Using a Polymer Stationary Phase

Routinely, for protein structure characterisation, the peptide mapping step involves separation of the protein fragments by means of reversed phase chromatography on silica C8 or C18 sorbents using ...

Pilot scale purification of phosphorthioate DNA oligonucleotides

The development of a single step chromatographic method for pilot scale purification of a synthetic phosphorothioate DNA 20-mer is described. This method is based on anion exchange chromatography ...

Pilot scale purification of synthetic DNA oligonucleotides

• One step purification of a DNA 20-mer • Gram scale purification capacity • Purification under denaturing conditions using aqueous buffers A single step chromatographic method for pilot scale ...

Proteome profiling of rat spermatozoa using three different LC-MS/MS configurations

The rat spermatozoa proteome was analyzed using the three preprogrammed configurations for the Ettan™ MDLC: high-throughput 1D-LC, online salt-step 2D-LC, and offline 2D-LC. The offline 2D-LC–MS/MS ...

Purification and chromatographic characterisation of an integral membrane protein

ÄKTAFPLC controlled by UNICORN™ software was used at cold room temperature for one-step purification and for characterisation of an integral membrane protein in the presence of non-ionic detergents. ...

Purification and renaturation of recombinant proteins produced in Escherichia coli as inclusion bodies

Expression of eukaryotic genes in the cytoplasm of Escherichia coli often leads to the production and accumulation of the recombinant protein (r-protein) to levels of total cell protein that are ...

Purification of (His)6-tagged proteins using HiTrap Chelating HP columns charged with different metal ions

Nickel is the first choice for metal ion according to the results in this investigation using these proteins, but to achieve optimal separation of (His)6-tagged proteins from cellular contaminants, ...

Purification of (His)6 -tagged recombinant proteins expressed as inclusion bodies in E. coli using a Ni2+ -charged HiTrap Chelating HP column

A chromatographic method for the purification of (His)6 -tagged recombinant proteins expressed as inclusion bodies in Escherichia coli is described. The inclusion bodies are solubilized in 8 M urea ...

Purification of a labile, oxygen-sensitive enzyme for crystallisation and 3D structure determination

A rapid purification procedure for deacetoxycephalosporin C synthase (DAOCS), an oxygen-sensitive enzyme involved in cephalosporin/cephamycin biosynthesis, was developed using ÄKTAFPLC. The aim was ...

Purification of a Phosphorylated PDGF alpha-Receptor Derived Peptide of High pH using Polymer Stationary Phase

Peptide pY574a is an 18 amino acid residue phosphorylated peptide constituting part of the intracellular domain of the PDGF a-receptor, which is involved in signal transduction. The peptide was ...

Purification of Amyloid-ß 1-42 at High pH using a Polymer Stationary Phase

Amyloid-b 1–42 is a peptide which is generated from a large transmembrane precursor protein by proteolytic cleavage. The peptide may polymerise to form rigid, linear, non-branching fibrils building ...

Purification of histidine-tagged Maltose Binding Protein with Ni Sepharose 6 Fast Flow - method optimization and scale-up

The work presented here describes the process optimization for the capture step for purification of MBP-(His)6 protein at high sample load using Ni Sepharose 6 Fast Flow. By reducing the ...

Purification of MBP-tagged proteins using new prepacked columns

Maltose binding protein (MBP) is beneficial as an affinity tag due to its ability to increase expression level and solubility of the fusion protein. The new MBPTrap™ HP 1 ml and 5 ml columns have ...

Purification of monoclonal immunoconjugates

This Application Note describes the use of several nonaffinity modes of chromatography for purification of immunoconjugates. Although individual monoclonal antibodies are very homogenous with respect ...

Purification of peptides from natural sources: Isolation and characterization of cholecystokin-58 (CCK-58) from pig intestine: ÄKTApurifier

A method is described for purifying a 58-residue molecular form of cholecystokinin, CCK-58, from porcine intestinal tissue using ÄKTA™ purifier system controlled by UNICORN ® . After extraction of ...

Purification of Strep(II)-tagged proteins using new prepacked columns

The Strep(II)-tag is ideal as an affinity tag due to the small size (8 aa) often making removal of the tag unnecessary. The new StrepTrap™ HP 1 ml and 5 ml columns have been specially developed for ...

Purification of Synthetic peptides by RPC; ÄKTAexplorer

A general strategy for purification of synthetic peptides was used for rapid method optimisation and scale up, using ÄKTAexplorer and UNICORN ™. The strategy is described in detail for the ...

Rapid development of a purification process for a recombinant antigen binding fragment expressed in E. coli

This Application Note describes a systematic approach to process development using ÄKTAexplorer with UNICORN™ control. The purification of a recombinant antigen-binding fragment (Fab), expressed in ...

Rapid optimisation and development of an automated two-step purification procedure for monoclonal IgG antibodies; ÄKTAFPLC

This application note describes the development and optimisation of an automated two-step purification process for mouse monoclonal IgG antibodies (MAbs) on ÄKTAFPLC. Cell culture supernatants ...

Rapid purification of GST-fusion proteins from large sample volumes

Expression of recombinant proteins or domains fused to Glutathione S-transferase (GST) using the pGEX vectors allows easy purification by affinity chromatography on Glutathione sepharose™ under mild ...

Rapid screening of a scalable, intermediate purification step for recombinant EGFP with HiTrap HIC Selection Kit

We describe using the HiTrap™ HIC Selection Kit expanded with Butyl Sepharose™ High Performance (as HiTrap Butyl HP) to screen seven prepacked 1 ml HIC columns with different hydrophobicities. The ...

Removal and purification of trypsin-like serine proteases

In the living organism, proteolytic enzymes (proteases) are produced to degrade and modify proteins. Serine proteases form the most common group of the proteolytic enzymes, and they are extremely ...

Reproducible and robust peptide separations using Ettan MDLC, a multidimensional liquid chromatography system

This application note demonstrates the reproducibility and robustness of Ettan MDLC for high-throughput one-dimensional (1D) LC analysis and two-dimensional (2D) LC analysis, and highlights the ...

Sanitization of ÄKTApilot with sodium hydroxide

Sanitization of ÄKTApilot™ was assessed by subjecting the regular sanitization procedure to microbial challenge tests. The system was infected with solutions containing three strains of bacteria that ...

Scaling up high performance chromatography on source media and FineLINE columns

This application note describes the unique separation performance achieved with SOURCE ™ media and their retained performance when scaling up to production scale with FineLINE columns. It stresses ...

Screening and optimization of the loading conditions on Capto S

Increasing expression levels of recombinant proteins and the need for improved productivity and overall process economy puts extra demand on the next generation of chromatography media for the ...

Snags with tags: Some observations made with (His)6 -tagged proteins

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Superdex 200 prep grade - unmatched resolution and speed

Ever since the introduction of Sephadex™, gel filtration has occupied a key position in the purification of biomolecules. In gel filtration biomolecules in solution are separated according to ...

The properties of Tricorn high performance columns: a verification report

This verification report documents the excellent properties of Tricorn™ as a high performance column for high resolution purification of proteins at laboratory scale. The report confirms that Tricorn ...

The Trap platform and Tecan automation – efficient solutions for screening proteins

Unattended affinity purification and buffer exchange on AKTAprime plus

We describe a new protocol for affinity purification followed by immediate buffer exchange on ÄKTAprime™ plus chromatography system. The protocol proved to be very effective when tested on three ...

Use of sodium hydroxide for cleaning and sanitizing chromatography media and systems

Sodium hydroxide is widely accepted for cleaning, sanitizing, and storing chromatography media and systems. The benefits of its use include efficacy, low cost, ease of detection, removal, and ...