His-Tag Protein Purification

His-Tag Protein Purification is a series of processes intended to isolate target protein from a complex mixture. A His-Tag is an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. Several metal cations have high affinities for imidazole, the function group of His-Tag. Transition metal imidazole complexes are well known for M2+ (M = Mn, Fe, Co, Ni, Cu). Nickel is a widely used metal ion in the purification of His-Tag protein.

Fig.1 Schematic diagram of His-Tag protein purification principle.

• Add His-Tag
  The addition of polyhistidine affinity tags to proteins can be accomplished in several ways. They are small in size, low in charge, and easily integrated into target genes by PCR or site-directed mutagenesis.
• Binding
  A crude solution containing the His-Tag protein is applied to the column and binds based on the affinity tag-matrix interaction.
• Washing
  Other proteins are washed away with the appropriate wash buffer. These buffers should already contain low molar amounts of imidazole.
• Elution
  Specifically bound protein is eluted from the column, usually by competitive binding of similar molecules, by protease cleavage of the tag, or by destabilization of affinity tag-substrate interactions, for example by changing pH. The pure eluate can then be collected in reaction tubes for further study.

• His-Tag is very small and does not affect the downstream application of the protein.
• The His-Tag at the N-terminal is compatible with the bacterial transcription and translation mechanism, which is beneficial to protein expression.
• The use of IMAC (Immobilized Metal Ion Affinity Chromatography) to purify the His-Tag fusion protein is more convenient ^[1].
• His-Tag has little effect on the properties of the target protein and will not form dimers.
• The immunogenicity of His-Tag is relatively low, and the purified protein can be directly injected into animals for immunization and antibody preparation.
• It can be combined with other affinity tags to form dual affinity tags, which can be applied to various expression systems.

• Target proteins with strong hydrophobicity can be purified in the presence of non-ionic surfactant.
• Inclusion body proteins can be purified under denaturing conditions.
• Affinity tags like His6 or GST paired with an immobilized ligand (e.g. Ni2+ agarose resin) can be used to specifically bind to a target protein complex in order to purify and identify it.
• Semi-quantitation with known His-Tag protein as standards.

Thermal Cyclers

Thermal Cycler is a laboratory apparatus that amplify segments of DNA using PCR. A thermal cycler is also commonly called a DNA Amplifier, PCR Machine, or Thermocycler. The cycle normally lowers or raises the block’s temperature in preprogrammed discrete steps. A thermal cycler is important for a laboratory dealing in molecular biology and gene cloning.

PCR Workstation / PCR Hood

A PCR workstation, also called a PCR hood, is a work space, enclosed on three sides, that provides a space for doing amplification of DNA and/or RNA. PCR hoods are used in biology and genetic labs so that there isn’t any cross contamination between samples. PCR workstations have no circulation, which helps to prevent contamination, and UV lights for sterilization.

PCR Consumables

PCR consumables generally involve pipettes, 100ul, 500ul and 1ml centrifuge tubes, 12 consecutive rows of tubes, and tube racks (96-well plates, etc.) for placing the centrifuge tubes. It is best to perform PCR experiments on ice, so you need to prepare an ice box. We use a wide variety of PCR consumables for you to choose from to meet your different needs.

Laboratory Rockers and Shakers

A rocker or shaker is a piece of laboratory equipment used to mix, blend, or agitate substances in a tube or flask by shaking them. These important tools are used for a variety of applications including cell culture, DNA extraction, mixing reagents, and staining gels and blots. A shaker contains an oscillating board that is used to place the flasks, beakers, or test tubes.

Protein Purification Columns

His-tag purification uses the purification technique of immobilized metal affinity chromatography, or IMAC. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid. The most common ion for his-tag purification of a recombinant protein is Ni2+, though Co2+, Cu2+, and Zn2+ are also used.

Pipettes and Pipette Tips

Pipette is a measuring tool that moves liquid from the original container to another container within a certain range. It is widely used in clinical diagnostic laboratories, biotechnology laboratories, pharmaceutical and chemical laboratories, environmental laboratories, and food laboratories. We provide a variety of pipettes for you to choose to use in a variety of experimental research.

Chromatography Fraction Collector

The fraction collector is typically a rotating rack that can be filled with test tubes or similar containers. If the chromatographic separation is not for pure chromatographic analysis, but for other spectral identification, or to obtain small-scale preparation of a small number of test samples, fraction collection is necessary, and fraction collector collection is ideal and accurate in operation.

STEMart provides you with a variety of His-Tag protein purification equipment to meet your various R&D and application needs. If you have any questions or requirements for His-Tag protein purification equipment, please feel free to contact us.

Reference

• Spriestersbach A, Kubicek J, Schäfer F, Block H, Maertens B. Purification of His-Tagged Proteins. Methods Enzymol. 2015; 559: 1-15. Doi: 10.1016/bs.mie.2014.11.003. Epub 2015 May 4. PMID: 26096499