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Cat. No.: HPRR-10 (for 10ml)

Cat. No.: HPRR-100 (for 100ml)

Cat. No.: HPRR-1k (for 1000ml)

Description

HisPrem™ His-tag Purification Resin (Reductant/Chelator-resistant), commonly known as nickel column, is a novel purification medium that is compatible with reducing agents and chelating agents, allowing for simple, fast, efficient, and highly specific purification of His-tagged proteins.

Recombinant proteins with 6 histidine residues (6xHis-tagged recombinant proteins) or proteins with more than 6 consecutive histidine residues are referred to as His-tagged proteins. When protein samples are passed through HisPrem™ His-tag Purification Resin (Reductant/Chelator-resistant), the histidine residues on the His-tagged recombinant protein specifically bind to the nickel ions in HisPrem™ His-tag Purification Resin (Reducing Chelating Type), while other proteins are not bound. After washing, the His-tagged recombinant protein can be eluted under non-denaturing conditions, thus achieving separation and purification.

This product exhibits strong affinity, high selectivity, and large binding capacity for His-tagged recombinant proteins, enabling the obtainment of highly pure His-tagged recombinant proteins. It can be used for simple, fast, and efficient purification of His-tagged recombinant proteins expressed in various systems, including bacteria, mammalian cells, insects, and baculovirus. The purified proteins can be used for structural and functional studies, antibody preparation, protein-protein interactions, protein-nucleic acid interactions, and other research applications.

HisPrem™ His-tag Purification Resin (Reductant/Chelator-resistant) utilizes a highly cross-linked 6% agarose gel matrix and employs optimized arm coupling and nickel chelation techniques. Compared to most commercially available Ni-NTA agarose or similar products, it exhibits significantly reduced nonspecific protein binding, excellent pressure tolerance, and highly stable nickel chelation. Unlike other products, it does not require reloading of nickel ions when reusing the purification medium because nickel ions are not easily lost.

Ni-NTA agarose and most similar nickel column products are not tolerant to common concentrations of reducing agents such as dithiothreitol (DTT) and β-mercaptoethanol, as well as common concentrations of chelating agents such as EDTA. However, HisPrem™ His-tag Purification Resin (Reducing Chelating Type) can withstand reducing agents like DTT and chelating agents like EDTA. It can tolerate up to 20 mM DTT as well as 20 mM EDTA. This provides the possibility for purification of protein samples that require the addition of reducing or chelating agents. This product is not compatible with 8 M urea and 6 M guanidine hydrochloride. For purification of inclusion body proteins using 8 M urea or 6 M guanidine hydrochloride for protein dissolution, it is necessary to remove urea and guanidine hydrochloride through dialysis before using HisPrem™ His-tag Purification Resin for purification of His-tagged proteins.

This product is pre-chelated with nickel ions and appears blue. The particle diameter of the gel is 45-165 μm. The maximum pressure tolerance is 0.025 MPa, approximately 5.8 psi. The recommended flow rate for protein purification using a fixed flow rate is 0.5 ml/min.

This product can bind His-tagged proteins with high capacity and high specificity. The maximum protein binding capacity is 20-30 mg protein/ml resin. The actual maximum binding capacity depends on the molecular weight of the His-tagged recombinant protein to be purified, with

 larger molecular weights corresponding to higher maximum binding capacities, and smaller molecular weights corresponding to lower maximum binding capacities. For a protein with a molecular weight of 50 kDa, the actual maximum purification capacity per ml resin is approximately 6-10 mg, while for a protein with a molecular weight of 100 kDa, the actual maximum purification capacity per ml resin is approximately 12-20 mg. However, for proteins with the same molecular weight, the maximum binding capacity may vary due to differences in protein characteristics.

This product is stored in 30% ethanol, with 50 ml gel and 50 ml liquid in a total volume of 100 ml. It is recommended to fully resuspend the gel before use.

Precautions

• Do not freeze this product at -20°C or lower temperatures.
• If the lysis buffer or nickel column equilibration buffer contains reducing agents such as DTT, it is recommended to wash the nickel column with 10-20 column volumes of non-denaturing lysis buffer (preparation method provided below) in advance to thoroughly remove any residual free nickel ions. Afterward, the column can be equilibrated with lysis buffer or equilibration buffer containing reducing agents such as DTT before continuing with the purification process.
• During the use of this product, the concentration of buffer reagents such as Tris, HEPES, MOPS, etc., should not exceed 100 mM. The concentration of EDTA should not exceed 20 mM. The concentrations of SDS and Sarkosyl should not exceed 0.3%. The concentrations of Triton, Tween, and NP-40 should not exceed 2%. The concentration of sodium deoxycholate and CHAPS should not exceed 1%. The concentration of histidine should not exceed 20 mM. The concentration of calcium ions should not exceed 5 mM. The concentrations of sodium and magnesium ions can go up to 2 M. The concentration of glycerol can go up to 50%. The compatibility of other reagents not mentioned can be referenced from the above-mentioned reagents, but experimental validation is still required.
• The gel should always be kept moist during storage and purification processes.
• We offer a range of empty columns for affinity chromatography in various sizes that are compatible with this product. If you are interested or have any specific requirements, please feel free to inquire.
• If centrifugation does not completely remove insoluble material from the protein sample, the sample solution can be filtered using a 0.45 μm filter membrane.
• Once protein samples are collected, it is recommended to complete the purification process as soon as possible and store them at 4°C or on ice to slow down protein degradation. To effectively inhibit protein degradation, an appropriate amount of a mixture of protease inhibitors can be added to the lysis buffer.
• If the desired purification results cannot be achieved using the conditions provided in this manual, try adjusting the concentration and/or pH of imidazole in the wash and elution buffers to optimize the results.
• If it is necessary to use denaturing agents for protein purification, we recommend using HisPrem™ His-tag Purification Resin (Denaturant-resistant) or HisPrem™ His-tag Purification Resin (Fast Flow, Denaturant-resistant).
• This product is intended for scientific research by professionals only and should not be used for clinical diagnosis or treatment, food, or drugs. It should not be stored in a regular household setting.
• For your safety and health, please wear lab attire and disposable gloves when handling.

Storage

Store at 4°C. Valid for at least one year.

Only for research and not intended for treatment of humans or animals

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SBS Genetech is a long-term sponsor of Cold Spring Harbor Laboratory