His tag protein purification tool - Huaqiang Electronic Network

His Tag Protein Purification Tool

Polyhistidine-tagged recombinant proteins expressed in E. coli and other prokaryotic systems are typically purified by immobilized metal ion affinity chromatography or referred to as IMAC. In this process, the support (usually a beaded agarose gel) Or magnetic bead particles) Derivatized with a suitable coupling reagent, such as nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA). These chelating groups will immobilize the desired divalent metal ion (for example) Nickel, copper, cobalt and zinc), then these metal ions are ultimately used as contact molecules and target molecules in the mixed protein. In choosing which ligand to use, you need to consider the binding capacity of the IMAC resin mainly depends on the nature of the protein being purified and the metal ions used in the purification process.

Highly purified His-tag fusion protein with NTA, IDA, and IMCA-type His-tag purified packing or prepacked column products

His-Tag affinity chromatography packing

His-Tag

Can interact with a variety of metal ions, such as

Ca2+

,

Mg2+

,

Ni2+

,

Cu2+

,

Fe3+

Etc., the principle is to use the characteristics of the surface of the protein to be adsorbed on the gel column, so as to achieve the purpose of separating and purifying the protein.

Ni2+

It is the most widely used in affinity purification experiments. Depending on the binding group,

Ni2+

Affinity chromatography columns can be divided into two categories -

Ni-IDA

with

Ni-NTA

.

Ni2+

There are six chelating prices, of which

Ni-IDA

Chelated the trivalent,

Ni-NTA

Chelated the tetravalent. and so

IDA

Load ratio

NTA

High under the same conditions

Ni-IDA

The concentration of imidazole at the time of elution is also higher than

Ni-NTA

. However, its weak binding force makes the metal ions easily leaching during the elution stage and tightly binds to the target protein, resulting in low protein yield, impure product and metal ion contamination. and

NTA

The particle size is uniform, the particle size is smaller, and the chelated nickel is more stable, can withstand higher reducing agent, makes the filler more stable, and the nickel ion is not easy to fall off.

His-tag is the preferred label for protein purification and its advantages are:

1.

N-

End

His-Tag

Compatible with the transcriptional translation mechanism of bacteria, which is beneficial to protein expression;

2.

use

IMAC

(immobilized metal ion affinity chromatography) purification

His-Tag

The fusion protein is easier to handle;

3.

His-Tag

Has little effect on the properties of the target protein itself, and does not change the solubility and biological function of the target protein itself;

4.

His-Tag

Very small, has no effect on the structure of the protein after crystallization of the fusion protein;

His-Tag

The immunogenicity is relatively low, and the purified protein can be directly injected into an animal for immunization and preparation of an antibody;

It is constructed as a parental tag with other affinity tags and can be applied to a variety of expression systems;

His-Tag

Fusion proteins are also available in a wide range of applications, either in the presence of nonionic surfactants or under denaturing conditions. The former is usually used to purify a hydrophobic protein of interest, while the latter is usually purified from inclusion bodies.

IMAC

It is relatively stable under normal conditions, but the presence of a chelating agent causes its metal ions to fall off. For example in

E.coli

Some non-specific weak chelating agents are commonly found in lysates, such as dicarboxylic acids in the tricarboxylic acid cycle. therefore,

E.coli

High specificity of metal chelators (usually present in the periplasm of bacteria) under certain high pressure conditions, resulting in

His-Tag

The purity and yield of the fusion protein purification is greatly reduced. So in the purification

His-Tag

In the experiment of fusion proteins, it is first necessary to remove the chelating agent.

The PurKineTM His-Tag Purification System is based on an innovative high-load IMAC matrix that ensures efficient purification of His-Tag protein from total lysate in a single step. Our patented metal ion chelation scheme makes it compatible: reducing agents (such as DTT), chelation metalloproteinase inhibitors (such as EDTA), and most buffer substrates and salt environments. PurKine product line

Available in many forms such as:

Free combination: chelating group (IDA, NTA or Super NTA), cross-linking medium (4% or 6% cross-linked agarose gel, magnetic beads), and metal ions (Ni, Co, Cu, metal-free IMAC) ), the patented assembly chelation formulation ensures maximum yield, stability and solubility of His-tagged proteins.