Includes ready-to-use pre-packed Ni spin columns and all required buffers for purification
Purification of ≥1 mg His-tagged protein per column in as little as 15 minutes
High specific binding of His-tagged proteins yielding purities of >95%
Strong nickel ion binding provides excellent resistance to EDTA and reducing agents. Compatible with commercially available detergent-based cell lysis reagents
Isolation and purification of His-tagged fusion proteins under native or denaturing conditions
Spin columns containing an affinity matrix for the small-scale isolation and purification of polyhistidine-tagged (His-tagged) fusion proteins. Immobilized Metal Affinity Chromatography (IMAC) purifications employing NEBExpress® Ni Spin columns can be performed under native or denaturing conditions, yielding highly pure target in a single protein purification step. This enables screening of expression conditions and streamlines the functional and structural characterization of the target protein.
Support Matrix: Spherical, agarose based microparticles ranging in size from 10-100 μm.
Figure 1: NEBExpress Ni Spin Column Quick Start Protocol
Figure 2: Purification of His-tagged fusion proteins using NEBExpress Ni Spin Columns
NEBExpress Ni Spin Columns were equilibrated and loaded with 500 μl of crude extract from E. coli containing a plasmid that expresses either His6-GluRS or His6-NDK fusion protein. Columns were washed, eluted and analyzed by SDS-PAGE to confirm low non-specific binding of extract proteins and high isolation of target. Marker (M) contains Color Prestained Protein Standard (NEB #P7719), Lane 1: GluRS protein load, Lane 2: GluRS flow through, Lane 3: GluRS 1st elution, Lane 4: GluRS 2nd elution, Lane 5: NDK protein Load, Lane 6: NDK flow through, Lane 7: NDK 1st elution, Lane 8: NDK 2nd elution.
This product is related to the following categories:
Isolation and purification of His-tagged fusion proteins under native or denaturing conditions as IMAC tolerates a wide range of conditions, including the presence of protein denaturants, reducing agents and detergents.
High specific binding of His-tagged proteins from various expression systems yielding milligram quantities of target protein with purities of >95%.
Can be used with common cell lysis reagents and a variety of buffer additives
Several E. coli proteins are capable of binding immobilized metal affinity matrices with moderate to high affinity. The most common contaminants include SlyD (28kDa), GlmS (67kDa), ArnA (74kDa) and carbonic anhydrase (25 kDa) (1). The named contaminants may be eliminated from the Ni column elution fraction by employing NiCo21(DE3) (NEB# C2529H) as the expression host and then performing a second binding step with chitin magnetic beads (NEB# E8036) (2).
Protein yield and purity are dependent upon the expression level, conformation and solubility characteristics of the recombinant fusion protein. For best results, estimate the expression level of the His-tagged protein of interest by first running a sample of the crude lysate on an SDS-PAGE gel.
Unlike most IMAC resins, the unique chemistry employed in NEBExpress™ Ni Resin provides chemical tolerance to chelating agents such as EDTA and reducing agents such as DTT. Please consult the chemical compatibility table for more information.
Polyhistidine tags do not typically compromise the biological protein function and are not considered immunogenic, however if cleavage of the His-tag is necessary we recommend using TEV Protease (NEB# P8112).
Ni resin yields highly variable binding capacities dependent on the target and conditions. The binding capacity value (≥ 1 mg/column) for this product was determined by performing a mock purification from crude cell lysates expressing a His-tagged target protein.
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