His-tagged protein purification with Ni column, so simple!

Introduction

In 1975, Porath et al. proposed a new purification method - immobilized metal chelate chromatography. This method uses coordination-chelation interaction of metal ions (Ni2+, Cu2+, etc.) with residues on the surface of amino acids (such as the imidazolyl group of histidine) to purify proteins that have an affinity for metal ions.

The histidine tag is widely used because it has a small molecular weight and hardly interferes with the function, activity and structure of the target protein. Fixed metal ion affinity chromatography is the most common method for purifying histidine-tagged proteins.

Histidine-tagged protein purification tool

Welch Ni Tanrose 6FF(NTA)

The Ni Tanrose 6FF (NTA) media is an affinity chromatography media formed by chelating Ni2+ on a 6% highly crosslinked agarose gel with nitrilotriacetic acid as the ligands. Welch Ni Tanrose 6FF (NTA) has high purity, the combined capacity can reach ~40mg His tag protein/ml by controlling the reasonable Ni ion density, which can be used for purification of histidine-tagged (6xHis-tagged) proteins from various expression sources (such as E. coli, yeast, insect cells, and mammalian cells). NTA contains four chelating zones that bind Ni2+ better than the general tridentate chelating agent. 6xHis can be chelated with Ni2+, so that the His-tagged protein is bound to the Ni Tanrose 6FF (NTA) purification media, the unbound protein is washed, and the protein bound to the media is mildly eluted by a certain concentration of imidazole or low pH buffer, so as to obtain the target protein with high purity.

It has the advantages of high load capacity, good selectivity, easy regeneration and low cost.

PreCot Ni 6FF (NTA) is pre-packed Ni Tanrose 6FF (NTA) column (1ml and 5ml) that can be used to purify 6xHis-tagged protein using a syringe, peristaltic pump, or liquid chromatography system (such as AKTA or FPLC).

Application

Pre-Packed Column: PreCot Ni 6FF（NTA）5ml

Sample: His-tagged protein (e. coli expression)

Equilibrium solution A: 50mM tris-HCl, 0.5m NaCl, 20mM imidazole, pH8.0

Eluent B: 50mM tris-HCl, 0.5m NaCl, 0.5m imidazole, pH8.0

Flow rate: equilibrium and elution-1.0ml/min, sample loading-0.5ml/min

Note:

1-3: no imidazole in the sample and equilibrium solution

4-6: 20mM imidazole in sample and equilibrium solution

Since histidine and/or cysteine amino acid residues also exist in host proteins, other non-specific proteins and target proteins bind to metal ion affinity chromatography media together, resulting in low purity of purified samples. By increasing the imidazole concentration in the sample, histidine-tagged protein can be purified by Ni Tanrose 6FF (NTA) in one step to obtain a purified sample with a purity of more than 85%.

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