Choose the right purification media
Ni Sepharose™ 6 Fast Flow (FF) medium is ideal for:
1. Manual purification such as gravity-flow and batch purification
2. Expression screening in multiwell plates
3. Higher flow rates purification with a laboratory pump or chromatography system such as ÄKTAdesign™ systems from GE Healthcare
4. Easy scale-up
Ni Sepharose™ High Performance (HP) medium is designed to give:
1. More concentrated target protein
2. High performance purification
It is preferably used with a laboratory pump or a chromatography system such as ÄKTAdesign systems from GE Healthcare. |
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What you need to know about Histidine-tag protein purification
 Histidine-tag, most commonly known as "His-tag", is the most used tag world wide for tagging recombinant proteins. This tag is mostly used for facilitation of the purification of expressed recombinant proteins, but also for detection purposes.
Interest in using tagged proteins is increasing
It is likely that the use of recombinant tagged proteins will increase further since functional biology and structural genomics now are "booming". The Histidine-tag is small and does not, in most cases, interfere with the function/activity/structure of the protein, but to cleave off the tag is becoming more and more common. There is a need to increase the throughput in protein purification and using tags achieves this by adding simplicity to the purification protocol.
Immobilized Metal Affinity Chromatography for Histidine-tagged protein purification - popular and effective
The preparative purification of Histidine-tagged recombinant proteins by immobilized metal affinity chromatography (IMAC) is both popular and highly effective. IMAC exploits the ability of the amino acid histidine to bind chelated transition metal ions such as nickel (Ni2+), zinc (Zn2+) and copper (Cu2+). Histidine-tag is globally the most used tag, often found as six histidine residues in series. Histidine may also be present on the surface of many non-modified proteins. Of the metal ions used in this technique, nickel (Ni2+) has generally been proven to be the most successful.
To meet the need of improved products for Histidine-tagged protein purification, GE Healthcare has developed a broad range of IMAC products to meet its customers' requirements from the new end-user to the most experienced one.
Our new pre-charged Ni Sepharose media simplifies the purification procedure to three simple steps: Bind! Wash! Elute!
1. Bind your Histidine-tagged protein on the selected Ni Sepharose product
From the crude lysate containing your Histidine-tagged protein of interest, you may filter or load directly your sample on your column.
Alternatively for Ni Sepharose 6 Fast Flow, you may use the optimized batch/gravity flow protocol for simple protein purification without using a chromatography system.
The protein of interest will show strong binding affinity to the immobilized Ni2+ ions of the Ni Sepharose media – regardless whether your conditions are native or denaturing- whereas proteins showing next to none interaction for the Ni2+ ions will simply be washed away. Though, be advised that the binding capabilities are protein-dependant, thus binding, washing and eluting conditions are to be adjusted carefully for every single purification protocol.
2. Wash to remove most of the undesirable impurities
Although most impurities will probably be washed away during the first step, exposed histidine groups from other native proteins may also bind to the column. Therefore, the washing step conditions (e.g. buffer components concentrations, flow rate) are to be adjusted carefully so only the protein of interest remains on the column whereas the undesirable impurities are washed away.
3. Elute to recover the purified Histidine-tagged protein
To recover your purified protein fractions, competitive interactions with imidazole or lowering the pH of your elution buffer are the most common methods. Imidazole competitively interacts with immobilized Ni2+ ions to reverse the binding of the protein of interest to the Ni Sepharose media, whereas pH lowering affects the ionic interactions in between Ni2+ ions and the protein of interest. As imidazole might have to be removed by buffer-exchanging methods following purification, pH lowering can be really harsh, especially for pH-sensitive proteins.
GE Healthcares’ Ni Sepharose™ provide you with the highest binding capacities, yields and purification quality available on the market today with negligible leakage of Ni2+ ion, thus reducing your overall purification medium and buffer consumption. In addition, our product range offers both loose purification medium pack size and convenient pre-packed columns to meet your specific requirements.
In addition with our commitment to offer you the best products for Histidine-tagged protein purification, we invite you to consult our Chromatography Handbooks collection for further details about protein purification basics and strategies.
Customer feedback on Ni Sepharose 6 Fast Flow.
Consult the product selection guide to select the best product for your Histidine-tagged protein purification needs.
Find out how our Histidine-tagged protein purification products compare to other suppliers.
See our excellent track record as a supplier for the research community and biopharmaceuticals manufacturing processes. | 
