Utilizes ni nta nickelchelating resin and is supplied with native and denaturing buffers for efficient purification of recombinant proteins under different conditions histagged protein purification, protein and peptide purification, protein sample preparation and protein purification, proteins, expression, isolation and analysis. Data file 18117440 ae tagged protein purification ni. Ni nta column preparation resuspend ninta agarose slurry in a bottle container. They are not to be used for human diagnostic or drug purposes. Ninta spin kit handbook 012008 5 product use limitations qiaexpress products are developed, designed, and sold for research use. Can anyone help me with a protocol to stripoff ni from. Oem manufacturer in china, for ni nta agarose, deae agarose. Qiagen enables nucleic acid purification fp search. Ninta magnetic agarose beads are supplied as a 5% vv suspension in.
Ninta agarose is an affinity chromatography matrix for purifying. Nta binds metal ions tightly, allowing use of stringent washes. Ninta agarose is a nickelcharged affinity resin that can be used to purify recombinant proteins containing a polyhistidine 6xhis sequence. Qiagen ni nta spin kit, 16 to 24m bead, up to 300gspin column binding capacity, cell lysate start material, automated processing, macroporus silica support, up to 95% homogeneity in one step, for fast, smallscale purification of 6xhis tagged proteins, ideal for proteomics application, includes 50 ni nta spin columns, reagents, buffers. Proteins bound to the resin can be eluted with low ph buffer or competition with imid azole or histidine. The ni nta resin uses nitrilotriacetic acid nta, a tetradenate chelating ligand, in a highly crosslinked 6% agarose matrix. Purification of histagged proteins by imac is based on the affinity of histidine residues for immobilized metal ions e. Nta agarose enables efficient immobilizedmetal affinity chromatography imac using gravityflow chromatography. Ninta purification system thermo fisher scientific. Let the resin settle by gravity and gently aspirate the supernatant. You can imagine what will happen when loading your his tag onto a column that has free nickel present. Ni nta agarose 10 ml r90101 ni nta agarose 25 ml r90115 ni nta agarose 100 ml r90110 system components the ni nta purification system components are listed in the following table and include enough resin, reagents, and columns for six purifications. Ni nta column preparation resuspend ni nta agarose slurry in a bottle container. The ni nta resin can be used to purify 6x his tagged proteins under native and denaturing conditions.
Dear all, i am using ni nta agarose qiagen for purification of. High quality ninta products 80 mgml protein cube biotech. A handbook for highlevel expression and purification of 6xhistagged proteins. Check that the resin is contained in the narrow part of the column body before. Purecube ni nta agarose was exposed to 5mm dtt for 1 h a.
Cellfree dna dna clean up genomic dna microbial dna plasmid dna rna. Gfp was spiked into li lysates and purified on a 1 ml purecube cartridge filled with purecube 100 ni nta agarose at flow rates from 1 to 7 mlmin, and in a gravity flow batch procedure. The system is designed around the high affinity and selectivity of ni nta agarose. Ninta magnetic agarose beads under native conditions. Can anyone help me with a protocol to stripoff ni from used ni nta agarose for gravityflow chromatography. This technique makes use of the affinity of multiple histidine residues for nickel ni. Consequently, commercial ninta resins cannot capture a large number of his. Nta occupies four of six ligand binding sites of the nickel ion, leaving two sites free for interaction with the his 6tag. Ninta superflow is comprised of ninta coupled to superflow resin. Ninta resin, a 6% agarose imac resin for his tag protein.
Ail group, gre, university of dundee 2014 purification of histagged proteins materials ni nta agarose qiagen buffers lysis buffer. For more details of the advantages of biorobot systems see the. The resulting medium selectively retains histidinetagged recombinant proteins, allowing them to be purified from cellular contaminants or cell free systems. Ni nta agarose beads are widely used for protein purification due to its high affinity and selectivity for recombinant fusion proteins that are tagged with six tandem histidine residues. Dear all, i am using ninta agarose qiagen for purification of histagged proteins by. This resin consists of crosslinked agarose derivatized with nitrilotriacetic acid nta and provides good properties working in native or denaturing conditions. Ni nta is designed for high quality purification of 6xhistagged recombinant proteins from bacteria, insect and mammalian cells. Ac501 purification histagged proteins nickel nta agarose. The beads are incubated with cell lysate containing histagged protein and then magnetically separated from the supernatant manually or through automation usi. Can anyone help me with a protocol to stripoff ni from used ninta. Purification of 6xhistagged proteins by ninta affinity chromatography, however, can be performed under native or denaturing conditions and is not affected by problems arising from protein insolubility. Nitrilotriacetic acid nta is the aminopolycarboxylic acid with the formula nch 2 co 2 h 3.
Qiagen ez1 advanced xl nucleic acid automated purification. Ni nta agarose qiagen 1 ml column with luer lock on both ends mobitec. How imidazole can be washed off the ninta agarose beads. This resin can recover histagged proteins from a variety of expression systems such as baculovirus, yeast, mammalian and bacterial cells. Ninta superflow cartridge handbook the wolfson centre for. Histidine residues on the tag, connected via a short linker to the c or nterminus of the protein, bind to the niions. Qiagen ni nta hissorb plate, 96well, transparent, 5 ni nta coated, histidine residued epitope detected, plate based format, manual instrument, ligand on plate, multiwellbased assay system, enhanced signaltonoise ratio and reproducibility, reduced optimization, for assays using 6xhistagged biomolecules. Thermo scientific pierce ni nta magnetic agarose beads provide a fast, convenient method for purification of histagged recombinant proteins. Protein purification with the ninta protein purification system. Highlyefficient purification of native polyhistidinetagged. Ni nta agarose is a nickelcharged affinity resin that can be used to purify recombinant proteins containing a polyhistidine 6xhis sequence. Ninta superflow biorobot handbook supplied with the columns or contact one of the qiagen.
Ni nta agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a his tag. Proteins bound to the resin can be eluted with low ph buffer or competition with imidazole or histidine. Nickel nta agarose beads are provided readytouse for rapid purification of histagged proteins under native or denaturing conditions. Purecube ni nta agarose is robust against oxidation and regenerable. Histidine residues in the his tag bind to the vacant positions in the coordination sphere of the immobilized nickel ions with high specificity and affinity. Ninta superflow cartridges or qiagen products in general, please do not. Component composition quantity ni nta agarose 50% slurry in 30% ethanol 10 ml. Ni affinity column can effectively purify extract even just one column. Qiagen ni nta agarose, 25ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start material, nickelcharged resin, 2. Ninta superflow cartridge handbook 032007 7 introduction qiagen ni nta superflow cartridges are prefilled with 1 ml or 5 ml ni nta superflow and are ready to use for purification of 6xhistagged proteins using a syringe, peristaltic pump, or liquid chromatography system such as the aktadesign or fplc system. Ni sepharose high performance consists of highly crosslinked 6% agarose beads to which a chelating group has been immobilized. High dynamic binding capacity of purecube 100 ni nta agarose.
The metal ions are immobilized on chromatographic matrices by a chelating ligand, most commonly nitrilotriacetic acid nta or iminodiacetic acid ida. Proteins bound to the resin may be eluted with either low ph buffer or by competition with imidazole or histidine. This offer is valid until december, 2019 and only when order is placed online through qiagen web shop, your organizations purchasing system, your sales rep, or by email using the cyber19 promo code. Ninta superflow biorobot handbook supplied with the columns or contact. Ninta resin for recombinant proteins carrying a small affinity tag consisting of. For manual or automated purification of histagged proteins pdf 111kb. Ni nta resins are commonly chosen for histaggedprotein purification because. Mar 27, 2007 the nickel nitrilotriacetic acid nta is a qiagen patented resin which offers affinity purification of 6xhistagged proteins expressed as recombinant proteins from sources like e. The target protein structure may also add to purification difficulties by blocking the his.
The ni nta resin is compatible with native or denaturing conditions and can be used in multiple formats, including conventional gravityflow chromatography, spin column and fplc. The ni nta resin uses nitrilotriacetic acid nta, a tetradenate chelating ligand, in a highly cross linked 6% agarose matrix. Manual purification of 6xhistagged proteins from e. Ni nta agarose consists of the chelating ligand nitrilotriacetic acid nta immobilized on 6 % crosslinked agarose beads that are suitable for batch binding, gravity flow, and fplc columns. The cleared lysate was applied to a nickel column ni nta agarose, qiagen, washed with 2 m salt and 20 mm imidazole, and eluted with 250 mm imidazole contained buffer 50 mm hepes ph 7. Ni nta agarose 500ml nta agarose purification from left to right 1. Ni nta agarose uses nta which represents the most commonly used chelating ligand in imac.
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