AminTRAP HIS Prepacked Column

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2 INDEX Ordering Information... 3 Intended Use... 3 Product Description... 3 Purification Procedure... 4 Sample Preparation... 5 Sample Purification... 6 Analysis... 6 Regeneration Procedure... 6 Use and Use Restrictions: The Products are sold, and deliverables of any services are provided for the purposes of the buyer s internal in vitro research, development or educational use only, not for in vivo, or any therapeutic or diagnostic use, nor for resale, or for providing services or any other commercial use of any kind, including without limitation, for any transfer in any form (including as part of a kit) to a third party; for analysis or reverse engineering of the Product or for manufacturing. Products should only be used in accordance with any safety data sheets, guidance or protocols that we issue from time to time and are available for download from our Site. Protective clothing should be used at all times when handling our Products. Safety datasheets relating to all Products are available for download from the Site or upon request. Expedeon grants no other license or rights under any intellectual property in respect of Products or services deliverables and in particular grants no license to use any Product or deliverables for any commercial purposes. Sale of Products or service deliverables by us or our authorised distributors are expressly conditional upon the customer s agreement with these restrictions, which the customer gives upon placing an order for Products or deliverables. If you wish to use any Product or deliverables for any purpose other than your own internal research as described above, you will require an additional licence from Expedeon. Please contact licensing@ expedeon.com.

3 ORDERING INFORMATION PRODUCT SIZE CAT. NO. 5x1ml 5x1ml TNN x5ml 5x5ml TNN50005 INTENDED USE This product is intended for research use only. Not intended for any animal or human therapeutic or diagnostic use. PRODUCT DESCRIPTION is a chromatography medium for purify 6xHistagged proteins expressed in series of expression vectors, such as E.coli., yeast, insect cells and mammalian cells. Ni-NTA resin consists of 90µm beads of highly cross-linked 6% agarose, to which Nitilotriacetic acid (NTA) has been coupled. The chelating group has then been charged with nickel ions (Ni2+). This form is very stable octahedral structure of nickel ions in the center, which can protect the nickel ions from attack of the competitive small molecule. The structure of Ni-NTA is compatible with a certain concentration of reducing agents, denaturing agents, detergents and other additives. ITEM Column size Matrix Binding capacity DESCRIPTION cm (1 ml); cm (5 ml) Highly cross-linked 4% agarose >40mg 6xHis-tagged protein/ml medium Particle size (μm) Maximum pressure Storage Solution 0.3 Mpa, 3 bar 1x PBS containing 20% ethanol Table 1. Characteristics of NI-NTA Resin is one of a range of prepacked, ready-to-use columns for affinity chromatography. It is packed with Ni-NTA resin and available in 1ml and 5ml. have a standard interface and can be adapted to all kinds of chromatography system, such as ÄKTA. It is fast, simple and easy to use. 3

4 REAGENT Reductants Denaturants Detergents Other additives Buffer Table 2. Chemical compatibilities of Ni-NTA Resin STABILITY 5 mm DTE 0.5-1mM DTT 20 mm β-mercaptoethanol 5 mm TCEP 10 mm reduced glutathione 8 M urea 6 M Gua-HCl 2% Triton X-100 (nonionic) 2% Tween 20 (nonionic) 2% NP-40 (nonionic) 2% cholate (anionic) 1% CHAPS (zwitterionic) 500 mm imidazole 20% ethanol 50% glycerol 100 mm Na2SO4 1.5 M NaCl 1 mm EDTA 60 mm citrate 50 mm sodium phosphate, ph mm Tris-HCl, ph mm Tris-acetate, ph mm HEPES, ph mm MOPS, ph mm sodium acetate, ph 4 PURIFICATION PROCEDURE 1. Buffer preperation The basic principle of the following recommended buffer and other buffer is low concentration of imidazole in Lysis and wash buffer and high in elution buffer. Water and chemicals used for buffer preparation should be of high purity. It is recommended filtering the buffers by passing them through a 0.22μm or 0.45 μm filter before use. can be used for the his-tagged protein purification under native conditions and denaturing conditions, which need different buffer. See table 3 and table 4. NAME VOLUME INGREDIENTS Lysis Buffer Wash Buffer Elution Buffer 50 mm NaH2PO4 (7.80 g NaH2PO4 2H2O) 300 mm NaCl (17.54 g NaCl) 10 mm imidazole (0.68 g imidazole) Adjust the buffer ph to 8.0 with NaOH solution 50 mm NaH2PO4 (7.80 g NaH2PO4 2H2O) 300 mm NaCl (17.54 g NaCl) 20 mm imidazole (1.36 g imidazole) Adjust the buffer ph to 8.0 with NaOH solution 50 mm NaH2PO4 (7.80 g NaH2PO4 2H2O) 300 mm NaCl (17.54 g NaCl) 250 mm imidazole (17.0 g imidazole) Adjust the buffer ph to 8.0 with NaOH solution Table 3. Recommended buffer for HIS-Tagged protein purification under native conditions 4

5 NAME VOLUME INGREDIENTS Lysis Buffer 8 M Urea ( g urea) 100 mm NaH2PO4 (15.60 g NaH 2 2H 2 O) 100 mm Tris HCl (15.76 g Tris HCl) Adjust the buffer ph to 8.0 with HCl solution Wash Buffer Elution Buffer 8 M Urea ( g urea) 100 mm NaH 2 (15.60 g NaH 2 2H 2 O) 100 mm Tris HCl (15.76 g Tris HCl) Adjust the buffer ph to 6.3 with HCl solution 8 M Urea ( g urea) 100 mm NaH 2 (15.60 g NaH 2 2H 2 O) 100 mm Tris HCl (15.76 g Tris HCl) Adjust the buffer ph to 4.5 with HCl solution Table 4. Recommended buffer for HIS-Tagged protein purification under denaturing conditions SAMPLE PREPARATION 1. Recombinant native protein expressed in E.coli or yeast Single colonies were cultured in LB medium. According to the instruction, adding the inducers for a period of time. Harvest cells from an appropriate volume of bacterial culture by centrifugation at 7,000rpm for 10-15min at 4ºC. Discard supernatant and determine weight of pellet. Resuspend pellet in 1:10 ration (w/v) in lysis buffer and add lysozyme (0.20.4mg/ml cell paste, if the host cell containing plyss or plyse, it can be without lysozyme) and PMSF (1mM/ml cell paste). If high concentration of cell suspension, it is consider to add 10μg/ml RNase A and 5μg/ml DNase I. Sonicate the cell suspension/lysate on ice. Centrifuge the homogenized lysate at 10,000rpm for 20min at 4ºC to clarify sample. Save supernatant. 2. Native protein expressed in yeast, insect or mammalian cells Harvest the cells from an appropriate volume of culture by centrifugation at 5,000rpm for 10-15min at 4ºC. Save supernatant. If the supernatant without EDTA, histidine and reductant, it can be purified directly, otherwise it need dialysis to 1XPBS under 4ºC. For a large volume of supernatant, it need precipitation by adding ammonium sulfate and dialysis to1xpbs under 4ºC. 3. Inclusion bodies from E.coli Harvest cells from an appropriate volume of bacterial culture by centrifugation at 7,000rpm for 10-15min at 4ºC. Discard supernatant and determine weight of pellet. Resuspend pellet in 1:10 ration (w/v) with Lysis Buffer. Sonicate the cell suspension/lysate on ice. Centrifuge the homogenized sample at 10,000rpm for 20min at 4ºC to pellet the inclusion. Resuspend pellet in 1:10 ration (w/v) with denaturing Lysis Buffer (containing 8M urea). Sonicate, as needed, to redissolve the pellet. Analyze the concentration of the target protein and continue with purification protocols under denaturing conditions. 5

6 SAMPLE PURIFICATION Fill the syringe or pump tubing with binding buffer. Remove the stopper and connect the column to the syringe (with the provided connector), or pump tubing, drop to drop to avoid introducing air into the column. Remove the snapoff end at the column outlet. Wash the column with 10 column volumes of binding buffer at 1 ml/min or 5 ml/min for 1 ml and 5 ml column respectively. Apply the sample, using a syringe fitted to the connector or by pumping it onto the column. Wash with 5 to 10 column volumes of binding buffer or until no material appears in the effluent. Elute with 5 column volumes of elution buffer. Other volumes may be required if the interaction is difficult to break. ANALYSIS Identify the fractions containing the 6XHis-tagged protein. Use UV absorbance, SDS-PAGE, or western blot. REGENERATION PROCEDURE A column used to purify protein from cell extract usually contains some soluble substances and cell debris that are nonspecifically absorbed onto the matrix. Cleaning-in-Place eliminates material not removed by regeneration and prevents progressive buildup of contaminants. If the column is to be reused, these contaminants should be cleaned from the column, as they were not completely removed during the sample clarification steps. Remove the strong hydrophobic binding protein, lipoprotein and lipid: Wash the column using 5-10 column volumes 30% isopropanol contacting for 1520min. Or you can choose the 2CV acidic or alkaline solution containing detergents, for example, 0.1 M acetic acid solution contains % non-ionic detergent, contacting for 1-2 hours. Finally wash the column with 10CV distilled water. Remove the proteins combined with ion interacting: Wash the column with 1.5M NaCl solution contacting for 10-15min. Finally wash the column with 10 column volumes distilled water. 6

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