If the ninta agarose changes from light blue to brownishgray, the. Qiagen ni nta agarose, 100ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start material, nickelcharged resin, 2. Gfp was spiked into li lysates in defined quantities 10, 5, 2. Nitrilotriacetate nickel an overview sciencedirect topics.
Purification of histagged proteins materials ni nta agarose qiagen buffers lysis buffer. The spin columns are supplied with a resin bed volume of 0. How imidazole can be washed off the ninta agarose beads. Sample to nigt continued ordering information qiagen promotion march 2017 until march 24, 2017 you can save 2 on selected assay and sample prepration products when you order 4% through your. The ni nta resin uses nitrilotriacetic acid nta, a tetradenate chelating ligand, in a highly cross linked 6% agarose matrix. Cgi114 protein concentrations were measured by absorbance at 280 nm mw 26,275. The next day thaw your frozen sample and carry it through to step 5.
Histagged protein purification under denaturing conditions prep. Ni nta agarose 10 ml 10 ml disposable columns, 5 ml bedvolume 5 5 disposable columns, 1 ml bedvolume 5 5 frits for 5 ml 10 10. This manual is supplied with the following products. Original manufacturer offers bulk volume of deae agarose, ni nta agarose, citacron blue agarose, cobalt nta agarose, deae cellulose de52, magnetic agarose beads and plain agarose beads. Ninta magnetic agarose beads are supplied as a 5% vv suspension with a binding capacity of 300 g protein per ml of suspension for 6xhis.
The size of the used agarose resin beads or magnetic beads influences the flow rated and the protein yield. Ni nta agarose qiagen 1 ml column with luer lock on both ends mobitec 10 ml luer lock syringe merck eurolab buffer composition equilibration buffer 20 mm trishcl, 200 mm nacl. The potential for unrelated, nontagged proteins to interact with the ninta resin is usually higher under. Ninta superflow cartridge handbook 032007 7 introduction qiagen ninta superflow cartridges are prefilled with 1 ml or 5 ml ninta 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 nta column preparation resuspend ni nta agarose slurry in a bottle container. Add 6 ml of sterile distilled water and resuspend resin. This is reflected in the larger number of proteins that appear in the first wash figure 23.
The potential for unrelated, nontagged proteins to interact with the ni nta resin is usually higher under native than under denaturing conditions. Ninta agarose and purification columns have the following specifications. A detailed protocol for a serological assay to detect sars. Ninta agarose purification of 6xhistagged proteins from e. The ni nta resin can be used to purify 6x his tagged proteins under native and denaturing conditions. If additional purification would be necessary, we recommend exclusion chromatography i. Pipet or pour 2 ml of the resin into a 10ml purification column supplied with the kit. Qiagen ninta agarose, 100ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start.
Dear all, i am using ninta agarose qiagen for purification of histagged proteins by gravityflow chromatography. Original manufacturer offers bulk volume of deae agarose, ni nta agarose. Expression and purification of proteins using 6xhistidinetag 4 1 introduction 1. The ninta resin uses nitrilotriacetic acid nta, a tetradenate chelating ligand, in a highly crosslinked 6% agarose matrix. During this step, the histagged target protein is purified under native conditions from the cleared e. The metal ions are immobilized on chromatographic matrices by a chelating ligand, most commonly nitrilotriacetic acid nta or iminodiacetic acid ida. Ninta magnetic agarose beads under native conditions. Qiagen ni nta agarose, 25ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start material, nickelcharged resin, 2. Most manufacturers recommend using 48 times before regeneration. Expression and purification of proteins using 6x histidinetag. Nickel nta agarose beads are provided readytouse for rapid purification of histagged proteins under native or denaturing conditions. Proteins bound to the resin can be eluted with low ph buffer or competition with imid azole or histidine. Ni nta agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a his tag.
Ninta superflow cartridge handbook 032007 7 introduction qiagen ninta superflow cartridges are prefilled with 1 ml or 5 ml ninta superflow and are ready to use for purification of 6xhistagged. Qiagen supplies the following ninta matrices for the purification of 6xhis tagged proteins. Nintaatto conjugates can be directly applied either to an sdspage gel or western blot membrane for fluorescence imaging, and have been successfully used in living cells. Dear all, i am using ni nta agarose qiagen for purification of histagged proteins by gravityflow chromatography. Purification of polyhistidinecontaining recombinant proteins. I dont if i am taking too high quantity of ninta, i use the one from qiagen, which typically has binding capacity of 50 mg per ml 2 wash the slurry with about 10 ml of water twice. Ni nta magnetic agarose beads are used for smallscale affinity purification as well as highthroughput screening of recombinant histagged proteins. If the resin changes from light blue to brownishgray the nickel has been lost or changed.
Ni nta agarose uses nta which represents the most commonly used chelating ligand in imac. Ninta agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a his tag. Perform routine affinity purification of histagged fusion proteins with thermo scientific hispur ninta resin, a highcapacity, highperformance nickelimac resin. Purification of histagged proteins by imac is based on the affinity of histidine residues for immobilized metal ions e. The ni nta resin uses nitrilotriacetic acid nta, a tetradenate chelating ligand, in a highly crosslinked 6% agarose matrix. Histidine residues in the his tag bind to the vacant positions in the coordination sphere of the. Ni nta can then be coupled to agarose resin or magnetic beads for imac immobilized metal chelate affinity chromatography.
Ninta purification system thermo fisher scientific. Excellent protein recovery with purecube 100 ni nta agarose. Check that the resin is contained in the narrow part of the column body before. Histagged protein purification under denaturing conditions. Ninta superflow columns have been specially designed and optimized for both manual applications and for automated largescale purification of 6xhistagged. Mar 27, 2007 the ni nta agarose resin is supplied as 50% slurry with ethanol. Jul 28, 2003 all chemicals are purchased from merck except for nintaagarose qiagen, glutathioneagarose sigma and coomassie brilliant blue g 250 serva. Find and order beads and products like this ni nta agarose on commandez abin4368201. Ninta agarose qiagen 1 ml column with luer lock on both ends mobitec 10 ml luer lock syringe merck eurolab buffer composition equilibration buffer 20 mm trishcl, 200 mm nacl. Histagged protein purification, protein and peptide purification, protein sample preparation and protein purification, proteins, expression, isolation and analysis. Equilibrate the ninta superflow resin by adding 600 l buffer npi10 to each well and apply a vacuum for approximately 2 min or until the buffer has been.
A handbook for highlevel expression and purification of 6xhistagged proteins. Ac501 purification histagged proteins nickel nta agarose. High dynamic binding capacity of purecube 100 ninta agarose. Histagged protein purification with protease inhibitor. Recovered gfp was quantified by absorption at 488 nm. Keep these sample independent though the rest of the protocol. 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 agarose is an affinity chromatography matrix for purifying recombinant.
Nta agarose enables efficient immobilizedmetal affinity chromatography imac using gravityflow chromatography. Ninta agarose purification of 6xhistagged proteins. Homogenizer, with 2 100 ml grad cylinders for rinsing ice bucket with powdered dry ice falcon 2059 tubes 14 ml scalpel and glass. Wash the column with 2 volumes of regeneration buffer 6 m guhcl, 0. Basel, switzerland, who have purified more than 100 different proteins on ninta resin, we recommend a maximum of 5 runs per column. Pierce ninta magnetic agarose beads consist of highly crosslinked agarose beads embedded with magnetite and a covalently attached tetradentate nitrilotriacetic acid nta chelator charged with. 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. Step 2a batch purification of histagged proteins from e. Over 20% more yield obtained with purecube ninta agarose. If the ni nta agarose changes from light blue to brownishgray, the following regeneration procedure is recommended. Component composition quantity ni nta agarose 50% slurry in 30% ethanol 10 ml. Ninta nickelnitrilotriacetic acid agarose resin qiagen, his bind resin novagen, emd millipore, or histrap columns ge healthcare may be used for purification of the expressed proteins. 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. Resuspend the ninta agarose in its bottle by inverting and gently tapping the bottle repeatedly.
The qiaexpressionist 062003 3 contents kit contents 7 storage conditions 8 technical assistance 9 product use limitations 9 product warranty and satisfaction guarantee 9 safety information 10. Plasmid dna purification genomic dna purification cat. This protein purification system is based on the remarkable selectivity of our unique. Ni nta uses the chelating ligand nitrilotriacetic acid nta coupled to a crosslinked 6% agarose resin that is suitable for use in batch and gravity flow applications applications. Given that this agarose is remarkably expensive i would like to reuse it.
Ni nta agarose, deae agarose, deae cellulose de52, cm. Ninta uses the chelating ligand nitrilotriacetic acid nta coupled to a crosslinked 6% agarose resin that is suitable for use in batch and gravity flow applications applications. Ninta can then be coupled to agarose resin or magnetic beads for imac immobilized metal chelate affinity chromatography. Allow the resin to settle completely by gravity 510 minutes or gently pellet it by lowspeed centrifugation 1 minute at 800.
Ninta 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. Ninta agarose 10 ml r90101 ninta agarose 25 ml r90115 ninta agarose 100 ml r90110 system components the ninta purification system components are listed in the following table and. This is a purification method to obtain functional his tagged protein. All chemicals are purchased from merck except for nintaagarose qiagen, glutathioneagarose sigma and coomassie brilliant blue g 250 serva. High quality ninta products 80 mgml protein cube biotech. Qiagen ninta agarose, 25ml, 45 to 165m bead, up to 50mgml binding capacity, cell. 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. Ninta resin for recombinant proteins carrying a small affinity tag consisting of. Ninta resin, a 6% agarose imac resin for his tag protein. The polyhistidine tag is the most popular affinity tag and typically consists of six consecutive histidine residues 6xhis. The ninta agarose resin is supplied as 50% slurry with ethanol.
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. Basel, switzerland, who have purified more than 100 different proteins on ni nta resin, we recommend a maximum of 5 runs per column. Nta chemistry minimizes metal leaching during purification and is compatible with up to 10 mm. Purification of histagged proteins under native conditions using genaxxon ni ida or ni nta agarose 2. Gfp was spiked into li lysates and purified on a 1 ml purecube cartridge filled with purecube 100 ninta agarose at flow.
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