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Support >  Applications >  Tagged_Protein_Purification >  His-Tagged_Miniprep

Tech Note: Ligation-Free ChIP-Seq Library Preparation

A Revolutionary Solution for Recombinant Protein Purification

Capturem His-Tagged Purification Kits

Overview

Recombinant protein production is an important part of many studies in nearly all research settings, from academic institutions to biopharmaceutical and agricultural industries. Having the right tools to produce and purify tagged proteins can make a big difference in the ability to efficiently obtain meaningful data and make the most of every experiment. While conventional methods require a lot of time and effort, Capturem His-Tagged Purification Kits mark the beginning of a protein purification revolution. By uniting speed, ease of use, and high capacity in one powerful system, protein work can move forward as never before.

Traditionally, the protein purification task has fallen to immobilized metal affinity chromatography (IMAC) resin columns, which provide high capacity but require several hours to complete, due to long equilibration and binding times, as well as slow diffusion of large molecules through the resin bed. Capturem His-Tagged Purification Kits make use of next-generation membrane technology to get around these bottlenecks, and upgrade high-purity, high-throughput purification with a five-minute, room-temperature protocol. Spin columns have been assembled with novel nylon-based membranes specifically modified to have a much higher protein binding capacity when compared to conventional membrane pores. These kits (Table I) are available in two formats, Miniprep, which accommodates up to 800 µl of clarified cell lysate, and Maxiprep, which accommodates up to 25 ml of clarified cell lysate or culture supernatant.

Comparison of Capturem His-Tagged Purification Miniprep and Maxiprep Kits
  Miniprep Kit Maxiprep Kit
Yield ~100 µg/column ~2.5 mg/column
Sample Volume up to 800 µl up to 25 ml
Preparation Time 5 min 15 min
Concentration of Eluted Protein ~0.3–1 mg/ml up to 4.5 mg/ml

 

Modified membrane pores

In addition to producing protein of high purity and yield, the speed of this protocol significantly reduces the possibility of protein degradation or loss of activity, thus giving downstream applications a higher probability of success. This convenient, powerful tool performs consistently across native and denaturing conditions, in the presence of additives (e.g., glycerol, TCEP, etc.), and when performing tandem purification.

Feature Advantage
5- to 15-min protocol performed at room temperature Save time and avoid the cold room
High ratio of bed volume to wash buffer volume (1:300) Higher purity eluates: small bed volume traps fewer contaminants; relative high amount of wash buffer (300x bed volume) allows for more thorough washing
Small bed volume More concentrated protein (~0.3 to 4.5 mg/ml)
Compatibility with mammalian and bacterial cell lysates Ability to easily work with different experimental systems
Short residence time Save time, reduce possibility of protein degradation or loss of activity; higher success in downstream applications
High performance under native or denaturing conditions, and in the presence of common additives Flexibility to work under a wide range of experimental conditions
Functionality with various lysis buffers Choice of cell lysis system

Exceptional performance with a wide range of experimental conditions

Purification of recombinant proteins may involve many factors that could prove problematic for traditional systems, including denaturing conditions and the presence of additives in the samples and/or buffers. His-Tagged Purification Miniprep Kits maintain high performance levels in the face of all these potential complications.

A variety of additives were tested at various concentrations during the purification of 6xhis-tagged GFPuv (~29 kDa). Protein purification was successful under the standard protocol (see Methods) in the presence of EDTA, beta mercaptoethanol (βME), TCEP, DTT, and glycerol. In all cases, the majority of bound protein was recovered in the first elution, with eluates ranging from 75 µg to >200 µg per column.

Excellent protein purification in the presence of many common additives

Purification of GFPuv in the presence of a wide range of additives, at varying concentrations. Additives were included in the sample, equilibration, wash, and elution buffers for each experiment. Samples were eluted twice with 300 µl elution buffer each time.

Purification of the same his-tagged protein was also compared under native and denaturing conditions. The standard protocol was applied, and the resulting gel imagery shows excellent purification for all samples, regardless of denaturing conditions.

Comparison of protein purification under native and denaturing conditions

Purification of GFPuv under native and denaturing conditions. Spin columns were loaded with 800 µl of cell lysate, and all steps were performed with appropriate buffers for native and denaturing conditions. 8 M urea or 6 M HNC(NH2)2 were included in appropriate samples.

Compatibility with lysis buffers from other vendors

This enhanced membrane technology offers the added convenience of compatibility with different lysis buffers. Cell lysates containing his-tagged protein were prepared with xTractor Buffer and two other lysis buffers from different vendors. No deviations were made from the standard His-Tagged Purification Miniprep Kit protocol for any of the samples. Eluates show a high level of compatibility across lysis preps.

Compatibility with choice of cell lysis system

spin column compatibility with various lysis buffers. Cell lysates prepared with different lysis buffers were all treated according to the standard protocol, and purified in duplicate.

Tandem purification of his-tagged proteins

This system was also tested for tandem purification of his-tagged proteins. A single spin column was loaded with ~600 µl of a protein cocktail. The standard purification protocol was applied, and the desired proteins were recovered at high yield and quality in a five-minute period.

Tandem purification of 6xhis-tagged proteins.

Purification of several 6xhis-tagged proteins. The spin column was loaded with a protein cocktail containing 6xHN-AcGFP (29.6 kDa), 6xHN-FLAG-Myc-DsRed (~31.5 kDa), 6xhis-AB2431 (~17 kDa), and 6xhis-Ubiquitin (10.7 kDa).

Summary

Capturem His-Tagged Purification Kits are a revolutionary solution for recombinant protein purification from mammalian and bacterial cell lysates. High-capacity membrane pores with chemically enhanced surface area take a significant step beyond the limitations of conventional resins. With an easy, no-waiting workflow that consistently performs at exceptional levels, this next-generation tool leaves the cold room behind and streamlines an incredible number of experiments for protein research. High yield and purity are maintained in the presence of additives and under denaturing conditions, when used in cooperation with different lysis buffers, and under the demands of tandem protein purification.

Methods

Spin columns were first equilibrated by addition of 400 µl xTractor Buffer to the column followed by centrifugation at 11,000 x g for 1 min. Equilibrated spin columns were then loaded with 400–750 µl of clarified lysate from cells expressing 6xhis-tagged fusion protein, and centrifuged at 11,000 x g for 1 min at room temperature. The bound protein was then washed with 300 µl wash buffer, followed by centrifugation. Elution was performed by addition of 300 µl elution buffer containing 500 mM imidazole, followed by centrifugation. The eluted fraction was analyzed by electrophoresis on a 4–20% SDS polyacrylamide gel to verify the purity of the protein. Gels were stained with coomassie blue and destained according to standard protocol. An expected band was observed at the ~29 kDa molecular weight corresponding to 6xhis-tagged GFPuv (unless otherwise specified).

Compatibility with various reagents was tested according to this standard protocol, with additives included in the sample, equilibration, wash, and elution buffers. In all cases, samples were eluted twice with 300 µl elution buffer each. EDTA was tested at concentrations of 1, 5, and 10 mM. βME was tested at 10, 20, and 30 mM. TCEP was tested at 1, 5, and 10 mM. Upon addition of 10 mM TCEP, a thick precipitate was observed in the lysate, most likely due to the proteins in solution. (The flowthrough lane shows very little protein present for this experiment.) This precipitate was not loaded on the column. DTT compatibility was tested at 1, 5, and 10 mM. Glycerol was tested at concentrations of 10% in the equilibration and wash buffers, and at 5% and 10% in the elution buffer.

Purification under native and denaturing conditions followed the standard protocol, but with 800 µl of cell lysate. All steps were performed with buffers for native and denaturing conditions. 8 M urea or 6 M HNC(NH2)2 were included in appropriate samples.

Lysis buffer compatibility was tested with lysates prepared using xTractor Buffer as well as two buffers from other vendors. Samples were purified in duplicate using the standard protocol.

Tandem purification of multiple his-tagged proteins was tested as per the standard protocol, with ~600 µl of a protein cocktail containing the following: 510 µl 6xHN-AcGFP lysate (29.6 kDa), 19 µl 6xHN-FLAG-Myc-DsRed (~31.5 kDa, 1 mg/ml), 10 µl 6xhis-AB2431 lysate (~17 kDa), and 62.5 µl 6xhis-Ubiquitin (10.7 kDa, 0.3 mg/ml).

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