Ni-IDA-Agarose

The effective way for purification of 6x Histidine-tagged recombinant fusion proteins

Biontex offers with its Ni-IDA-Agarose an effective way for purification of recombinant proteins fused with a 6x Histidine-tag.

The product consists of the tridentate chelating agent, iminodiacetic acid (IDA), covalently coupled to 4% cross-linked agarose beads.

Ni-IDA-Technology

Immobilized-metal affinity chromatography was first used to purify proteins in 1975. Porath et al. used the chelating ligand iminodiacetic acid (IDA), which was charged with metal ions like Zn²⁺, Ni²⁺ etc.

IDA is a tridental chelating agent, covalently coupled to 4% cross-linked agarose beads and loaded with Ni²⁺ ion, which selectively and tightly bind histidine residues:

Elution is possible by several techniques, including lowering the pH, or with the use of a metal complexing agent such as imidazole or EDTA in the buffer. There is a balance between the concentration of imidazole required to elute the His-tagged protein, and the amount needed to avoid non-specific binding of contaminants.

Generally a higher imidazole concentration in the Equilibration Buffer will give a higher purity of protein, although it will compromise the yield. If high yield is important, see "optimization of elution for maximum yield" below.

Why Ni²⁺ or Co²⁺?

The two metal ions have different binding properties. Cobalt has strict requirements for the spatial positioning of the histidine residues and will only bind histidines next to each other. Nickel does not have such a tight requirement and will bind histidine residues elsewhere in the protein. Therefore, Cobalt has a tighter and more specific binding property than Nickel. Generally, Nickel is used to bind more protein and better elution.

The down side is that Nickel may also bind to other host proteins with histidine repeats. Cobalt is used to bind less protein but can be more specific resulting in preps with less background. The down side is that yields will be lower.

A (nitriloacetic acid) supports?

The NTA has 4 sites and the IDA has 3 sites available for interaction with metal ions. Generally, the NTA offers tighter protein binding and less metal ion leaching during purification process and therefore can withstand harsher purification buffers. However, it is more difficult to elute protein with NTA support.

IDA has a higher metal leaching level and binding is softer than NTA but purification of protein is easier. IDA supports therefore can be recycled better and more so than NTA without loss of binding activity.

Highlights of Ni-IDA-Agarose

Widely used system
Simple purification procedure under either native or denaturing conditions
His-tag does not affect bioactivity of protein
Direct purification from crude bacterial lysates
Best purity of protein
pH stability of 3 – 13 (short term 2 – 14)
Binding capacity of up to 10 mg 6xHis-tagged protein per ml
Resin can be regenerated for multible uses
Extremely cost effective
Generic tool for protein expression, purification and detection: no need to produce individual antibodies to each protein of interest

Applications*

Production of antigens for in vitro diagnosis
Production of vaccines and adjuvants
Production of chemical intermediates
Antigen production for generation of antibodies
Generation of research reagents

* Important Information

Biontex Ni-IDA-Agarose is developed and sold for research purposes and in vitro use only. It is not intended for human therapeutic or diagnostic purposes. Appropriate care should be exercised when handling many of the reagents described in this publication.

Comparison with other providers of Ni-IDA-Agarose

Biontex Ni-IDA-Agarose

Vendor "A" Ni-IDA-Agarose

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12

Lanes:

1, 12: Molecular Weight Markers
2: Cell Free Extract (CFE)
3: CFE post Nickel-IDA-Agarose Binding

4 – 7: subsequent column washes
8 – 11: subsequent elute collections

Evaluation of Biontex Ni-IDA-Agarose for purification of His-tagged Fusion Protein m-pisA. m-pisA (wt) is the structural gene for the peptide piscicolin 126. An antibacterial peptide produced from Carnobacterium piscicola JG1264. Theoretical expected size of full-length protein is 21.5 kDa.

Protocol

A cell free extract (CFE) from the fermentation of E. coli AD494(DE3) containing pET32a m-pisA (wt) is applied to the resin. Binding is carried out at room temperature for 30 minutes with regular mixing. All column wash and elution steps are performed as follows; buffer is applied to the resin and the resin is resuspended and allowed to settle. After the resin has settled the buffer is removed from the column by gravity flow and collected for later analysis by SDS PAGE. All column washes and elutions are 2 column volumes (ie. 2 ml).
Electrophoresis: samples are then heated in SDS loading buffer containing β-mercaptoethanol for 10 minutes at 95°C and applied to a tris-tricine SDS PAGE gel. 3.9% stacking gel and 10% separating gel.

General Principle of Protein Purification

Sample Preparation
Biontex Ni-IDA-Agarose preparation
Sample Application
Washing
Elution
Clean-up and regeneration

For more details and specifications of resin see our Ni-IDA-Agarose Solutions Guidebook.

Optimization procedure of elution for maximum yield:

Example for a complete arrangement for column chromatography, with automatic collection of eluate in fraction collector. The simple mixing device illustrated using the magnetic stirrer shows how the gradient elution can be set up. This can be replaced with an automated mixer and pump with pre-programmable gradient formation in modern apparatus.

References

P. Hansen, G. Lindeberg: Journal of Chromatography 690, 155-159 (1995)
S. Roe: Protein Purification Methods – A Practical Approach 175–242. Edited by Harris, E. L. V. & Angal, S. Oxford University Press, New York (1993)
R.K. Scopes: Protein Purification – Principles and Practice, Springer – Verlag, New York (1994)
Jack et al.: Applied and Environmental Microbiology, Aug 1996, Vol 62, 8:2897-2903

Prices

Reagent	Order No.	Price
Ni-IDA-Agarose 5 x 1 ml For purification of Histidine-tagged fusion proteins	R010-02.5	128 €
Ni-IDA-Agarose 10 ml For purification of Histidine-tagged fusion proteins	R010-10.1	156 €
Ni-IDA-Agarose 50 ml For purification of Histidine-tagged fusion proteins	R010-50.1	446 €
Ni-IDA-Agarose 2 x 50 ml For purification of Histidine-tagged fusion proteins	R010-50.2	807 €