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Cat. No.: HPRFR-10 (for 10ml)

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

Cat. No.: HPRFR-1k (for 1000ml)

Description

HisPrem™ His-tag Purification Resin (Fast Flow, Reductant/Chelator-resistant), commonly known as a nickel column, is a novel purification medium that can withstand high flow rates and pressures. It is compatible with reductants and chelators, allowing for simple, fast, efficient, and highly specific purification of His-tagged proteins.

Recombinant proteins containing a 6xHis-tag or more consecutive histidine residues are commonly referred to as His-tagged proteins. When a protein sample solution passes through HisPrem™ His-tag Purification Resin (Fast Flow, Reductant/Chelator-resistant), the histidine residues on the His-tag of the recombinant protein selectively bind to the nickel ions in the HisPrem™ His-tag Purification Resin (Fast Flow, Reductant/Chelator-resistant), while other proteins do not bind. After washing, the His-tagged recombinant protein can be eluted under non-denaturing conditions, resulting in its separation and purification.

This product exhibits strong affinity, high selectivity, and large binding capacity for His-tagged recombinant proteins. It can be used to obtain highly pure His-tagged recombinant proteins from various expression systems, such as bacteria, mammalian cells, insects, and baculoviruses, in a simple, fast, and efficient manner. The purified proteins can be used for structural and functional studies, antibody preparation, protein-protein interactions, protein-nucleic acid interactions, and other research applications.

This product utilizes a highly cross-linked 6% agarose gel matrix and employs optimized arm chemistry and nickel ion chelation technology. Compared to most commercially available Ni-NTA agarose or similar products, it significantly reduces non-specific protein binding, has strong pressure tolerance, and exhibits excellent stability in nickel ion chelation. Unlike similar products, this product does not require reloading of nickel ions when reused because the nickel ions are not significantly lost during the purification process.

Ni-NTA agarose and the majority of similar nickel column products cannot tolerate common concentrations of reducing agents such as dithiothreitol (DTT) and β-mercaptoethanol, as well as common concentrations of chelators such as EDTA, in protein samples. However, this product can withstand reducing agents like DTT and chelators like EDTA. It can tolerate 20 mM DTT as a reducing agent and 20 mM EDTA as a chelator. This provides the possibility of purifying protein samples that require the addition of reducing agents or chelators. This product is not compatible with 8 M urea and 6 M guanidine hydrochloride. For the purification of inclusion body proteins, when using 8 M urea or 6 M guanidine hydrochloride to dissolve the protein, the urea and guanidine hydrochloride need to be removed by dialysis before using this product for His-tagged protein purification.

This product is pre-chelated with nickel ions, giving it a blue color, and the gel particles have a diameter of 45-165 μm. The maximum pressure tolerance is 0.3 MPa, equivalent to approximately 43.5 psi. When performing protein purification at a fixed flow rate, the maximum flow rate is 300 cm/h.

This product can bind His-tagged proteins with high capacity and specificity. The maximum protein binding capacity is 20-30 mg protein/mL gel. The actual maximum binding capacity depends on the molecular weight of the His-tagged recom

binant protein being purified, with larger molecular weights resulting in higher maximum binding capacities and smaller molecular weights resulting in lower maximum binding capacities. For a protein with a molecular weight of 50 kDa, the actual maximum purification capacity per mL of gel is approximately 6-10 mg, and for a protein with a molecular weight of 100 kDa, the actual maximum purification capacity per mL of gel is approximately 12-20 mg. However, for proteins with the same molecular weight, the maximum binding capacity may vary due to differences in the protein's inherent characteristics.

This product is stored in 20% ethanol, with 5 mL of gel and 5 mL of liquid in a total volume of 10 mL. It is advisable 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