Product name: Ni-TED Sepharose FF


English name: Nickel Tris(carboxymethyl)ethylenediamine NUPharose Fast Flow, Ni-TED NUPharose FF


Catalog number: NRPB58L, NRPB58S (prepacked column)


Specifications: 20 ml, 100 ml, 1L, 1 ml pre-packed column, 5 ml pre-packed column, special specifications customized


Transportation: 2 ~ 30℃, normal pressure, avoid light


Storage: 20% ethanol, 2 ~ 25℃


Shelf life: 5 years






Related introduction:


Ni-TED Sepharose FF is based on agarose microspheres, coupled with tricarboxymethylethylenediamine (TED) through activation, and then chelate Ni2+. Compared with iminodiacetic acid (IDA), Ni-TED Sepharose FF has good chelating agent, reducing agent, alkaline tolerance, such as samples containing 10-100mM EDTA or 10-100mM β- Mercaptoethanol or 5-20mM DTT can be purified normally; no nickel removal is required, and 1M NaOH can also be used for regeneration and cleaning.






Ni-TED Sepharose FF is mainly used to purify recombinant proteins with histidine tag (His-Tag). The purification principle is that the imidazole ring of the histidine tag of the recombinant protein can form a stable coordination bond with the transition metal Ni2+, so it can be specifically, firmly and reversibly adsorbed to the matrix that fixes these metal ions, combined with the His-Tag recombinant protein Ni-TED Sepharose FF is generally eluted by increasing the concentration of imidazole.






Technical index:


Matrix


6% agarose gel


Ligand


-N(CH2COOH)CH2CH2N(CH2COOH)2


Ligand Density


≥30 μmol/ml


Filler particle size


60~180 μm


Maximum flow rate


800 cm/h


Recommended flow rate


50-100 cm/h


pH stability


pH 3-12 (cleaning pH 2-14)


Back pressure resistant


0.3 MPa


Capacity


≥20 mg His-tag recombinant protein/ml






Instructions:


1. Column packing


(1) The temperature of all materials that need to be used should be the same as the temperature of the chromatographic operation, and the liquid is best to be degassed.


(2) Add distilled water to the lower end of the column to remove the air in the column, close the outlet of the column, and leave a small amount of distilled water in the column.


(3) When continuously pouring the agarose gel into the column, use a glass rod close to the inner wall of the column for drainage to reduce the generation of bubbles and allow the filler to settle naturally.


(4) The column pressure should not exceed 0.3 MPa. If the column pressure cannot be measured in the column packing system, the column should be packed at normal flow rate.


(5) The packed Ni-TED Sepharose FF column is equilibrated with 2-5 column volumes of the initial buffer solution, and the recommended flow rate is 100 cm/h. The equilibrated column can be used for the loading of His-Tag recombinant protein. And elution purification.


2. Sample loading


(1) Buffer selection: Generally, the binding buffer with pH 6-8 is used. Commonly used buffers include 10-00mM sodium phosphate buffer, 20-200mM Tris-HCl buffer, etc. Generally, 0.15-0.5M NaCl should be added to the buffer to eliminate ion exchange. When using Ni-TED Sepharose FF for the first time, it is recommended to use 50mM PBS (50 mM NaH2PO4, 0.5M NaCl, NaOH to adjust pH 7.4) as the binding buffer.


(2) Sample processing: The sample buffer should be consistent with the selected binding buffer. In order to ensure the consistency of the buffer, the bacteria can be broken in the binding buffer, or the pH can be adjusted to be consistent with the binding buffer, or exchanged to the binding buffer (common methods include dialysis, ultrafiltration, desalting column exchange, etc.), or use the binding buffer The solution is diluted 2-10 times and so on. The sample should be filtered with 0.45μm before loading the column.


3. Elution


(1) The most commonly used elution method for Ni-TED Sepharose FF is to increase the concentration of imidazole for elution.


(2) Imidazole is alkaline, and pH should be adjusted with HCl after preparation of the corresponding buffer.


(3) When using Ni-TED Sepharose FF for the first time, I don’t know the concentration of eluted imidazole. It is recommended to add 10mM, 20mM, 50mM, 100mM, 200mM, 500mM imidazole to the binding buffer, and the concentration ranges from low to high. Separately eluted and collected, and identified the eluted components by SDS-PAGE electrophoresis and other methods.


(4) If conditions permit, linear imidazole gradient elution can also be performed to determine the best elution conditions.


4. Cleaning in place


When the used chromatography column is blocked, the pressure increases, the flow rate slows down, and the capacity drops, it needs to be cleaned in place. The advantage of Ni-TED Sepharose FF is that it can be regenerated and cleaned with 1M NaOH without nickel removal, so the most commonly used method of cleaning: wash the used column with distilled water for 3-5 column volumes, and then wash with 1M NaOH3 -5 column volumes (hold time 0.5-1h), then wash with distilled water to neutral. Like other chromatographic media, in order to achieve better cleaning purpose, reverse cleaning can be used; if the heat source needs to be removed, it can be kept in 1M NaOH for a longer time (1-2h).


In addition to NaOH cleaning, strongly charged proteins can be cleaned with 1-2M NaCl; lipids, lipoproteins, and strong hydrophobins can be cleaned with 30% isopropanol (or 70% ethanol).

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