Product Class: Other

NEBExpress® Ni Spin Columns


Catalog #S1427

Product Introduction

  • 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

Product Information

Description

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:
Amylose Purification (MBP-tag),
Nickel Purification (His-tag),
Affinity Purification Products,
Protein Purification Products,
This product can be used in the following applications:
MBP Affinity Tag,
Fusion Protein Cleavage,
Pull Down Assays,
His-tagged Protein Expression & Purification,
Affinity Purification & Expression Tags,
Protein Purification,
Protein Analysis Tools

Properties & Usage

Storage Buffer

20% Ethanol

Support Matrix

Spherical, agarose based microparticles ranging in size from 10-100 μm.

Binding Capacity

Varies with target, ≥ 1 mg His-tagged fusion protein per column.

Features

  • 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

Product Notes

  1. Do not freeze.
  2. 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). 
  3. 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. 
  4. 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. 
  5. 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).
  6. 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. 

References

  1. Bolanos-Garcia et al (2006). BBA. 1760, 1304-1313.
  2. Samuelson, J (2016). GEN. 26-27.

Protocols, Manuals & Usage

Protocols

  1. NEBExpress® Ni Spin Columns Quick Start Protocol (NEB #S1427)
  2. NEBExpress® Ni Spin Column Reaction Protocol (NEB #S1427)
  3. His-tag removal from protein using TEV Protease

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. How much lysate can be loaded onto a single NEBExpress Ni Spin Column?
  2. Will extended binding increase the yield of the target protein in the eluate?
  3. What is the minimum recommended load volume?
  4.  Is it necessary to cap the column during each centrifugation step?
  5. Why is the liquid not completely removed during the centrifugation step?
  6. I’ve prepared my lysate with too much buffer and the target is very dilute, can I apply more lysate by repeating the load steps?
  7. How can I reduce contaminating proteins in a Ni spin column protein purification?
  8. What are the recommendations for pH based elution, as opposed to imidazole?
  9. How can I remove imidazole from a protein sample?
  10. What are to the advantages of performing purification under denaturing conditions?
  11. How can I determine if my target protein remains bound to the resin?
  12. Why is imidazole not necessary in the lysis/binding buffer? Why does the wash buffer contain only 5mM imidazole?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Other Products You May Be Interested In