Ninta column preparation resuspend ninta agarose slurry in a bottle container. Pressure, manualautomated processing, large scale, sepharose cl6b matrix, 100g to 100mg yield, 6xhis. For purification of histagged proteins by gravityflow chromatography. Structurally, it is a linear polymer consisting of alternating dgalactose and 3,6anhydrolgalactose units. If the resin changes from light blue to brownishgray the nickel has been lost or changed its oxidations state and will no longer bind his tagged proteins. This material has excellent handling properties for most scales of batch and column purification. Ninta spin kit handbook 012008 5 product use limitations qiaexpress products are developed, designed, and sold for research use. Ninta agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a his tag. Qiagen ninta agarose, 500ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start material, nickelcharged resin, 2. Component composition quantity ninta agarose 50% slurry in 30% ethanol 10 ml. Order gbiosciencess gsep ni nta agarose fast flow on.
Nta agarose enables efficient immobilizedmetal affinity chromatography imac using gravityflow chromatography. Gfp was spiked into li lysates and purified on a 1 ml purecube cartridge filled with purecube 100 ninta agarose at flow rates from 1 to 7 mlmin, and in a gravity flow batch procedure. When using 96well blocks, cover block with tape and vortex 6 times for 5 s each time on a low to medium setting. Ninta agarose provides ninta coupled to a sepharose cl6b support and offers high binding capacity and minimal nonspecific binding see figure onestep purification under native conditions. Dear all, i am using ninta agarose qiagen for purification of histagged proteins by gravityflow chromatography. Recovered gfp was quantified by absorption at 488 nm. For manual or automated purification of histagged proteins pdf 111kb. Qiagen ninta agarose, 25ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start material, nickelcharged resin, 2. They are used for immobilizing and purifying recombinant. Ninta can then be coupled to agarose resin or magnetic beads for imac immobilized metal chelate affinity chromatography. Histagged proteinsproduction and purification thermo. Over 20% more yield obtained with purecube ninta agarose.
Ninta agarose, ninta superflow manufacturersupplier. Ninta agarose 6 ff is ideal for purification of histagged proteins using immobilized metal ion affinity chromatography imac. Proteins bound to the resin can be eluted with low ph buffer or competition with imid azole or histidine. 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. This step may need to be optimized for different cell cultures and vortexers. The size of the used agarose resin beads or magnetic beads influences the flow rated and the protein yield. Top 451 ramsey road, shirley, ny 11967, usa 6366 hatton garden, fifth floor, suite 23, london ec1n 8le, uk usa. Ac501 purification histagged proteins nickel nta agarose.
This resin can recover histagged proteins from a variety of expression systems such as baculovirus, yeast, mammalian and bacterial cells. Proteins from any expression system can be purified under native or denaturing conditions. Performance characterization of hispur cobalt superflow. The system is designed around the high affinity and selectivity of ninta agarose. Ninta 100 agarose his tag protein purification cube. Add 6 ml of sterile distilled water and resuspend resin. Gfp was spiked into li lysates in defined quantities 10, 5, 2. Excellent protein recovery with purecube 100 ninta agarose. 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. Ninta agarose uses nta which represents the most commonly used chelating ligand in imac. Print bookmark share more for manual or automated purification of histagged proteins.
Ninta agarose purification of 6xhistagged proteins from e. Expression and purification of proteins using 6x histidinetag. Qiagen ninta agarose, 100ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start material, nickelcharged resin, 2. 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 introduction and general guidelines 11 the qiaexpress system 15 qiaexpress pqe vectors 15 qiaexpress pqetrisystem vector for expression in li, mammalian and insect cells 16.
Qiagen supplies the following ninta matrices for the. Pressure, manualautomated processing, large scale, sepharose cl6b matrix, 100g to 100mg yield, 6xhis tag, affinity chromatography purification, high. Protein purification with the ninta protein purification system. Date hs code description origin country port of discharge unit quantity value inr per unit inr nov 21 2016. Check that the resin is contained in the narrow part of the column body before opening the columns. Let the resin settle by gravity and gently aspirate the supernatant. The ninta resin is precharged and able to bind up to 50 mg of recombinant protein per 1 ml of resin. The ninta agarose resin is supplied as 50% slurry with ethanol. Wash the column with 2 volumes of regeneration buffer 6 m guhcl, 0. Most manufacturers recommend using 48 times before regeneration.
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 include enough resin, reagents, and columns for six purifications. G sepni nta agarose fast flow is specifically designed for the purification of recombinant proteins fused to the 6x histidine 6xhis tag. Ninta magnetic agarose beads are magnetic particles coated with ninta agarose affinity purification matrix. All chemicals are purchased from merck except for nintaagarose qiagen, glutathioneagarose sigma and coomassie brilliant blue g 250 serva. Cgi114 protein concentrations were measured by absorbance at 280 nm mw 26,275. Nickel nta agarose beads are provided readytouse for rapid purification of histagged proteins under native or denaturing conditions. For efficient immobilizedmetal affinity chromatography imac using gravityflow chromatography. The resin is specifically designed for the purification of recombinant proteins fused to the 6x histidine 6xhis tag expressed in bacteria, insects, and mammalian cells. 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. Sonicate or homogenize on ice to lyse cells 6 times for 10 s each time with 5 s pauses between. When packed into suitable columns or cartridges, resins such as ninta superflow agarose provide for purification of 1 to 80 milligrams of histagged protein per milliliter of agarose beads. Ninta agarose purification of 6xhistagged proteins from.
Please refer to the invitrogen pbadhis a, b, and c manual, and qiagen the qiaexpressionist both kept in the lab folder. Basel, switzerland, who have purified more than 100 different proteins on ninta resin, we recommend a maximum of 5 runs per column. If the ninta agarose changes from light blue to brownishgray, the following regeneration procedure is recommended. Expression and purification of proteins using 6xhistidinetag 4 1 introduction 1. How imidazole can be washed off the ninta agarose beads. Ninta column preparation resuspend ninta agarose slurry in the bottled container. Manual purification of 6xhistagged proteins from e. Compared to cobalt and other ligands used for imac, nickel provides greater capacity for. Given that this agarose is remarkably expensive i would like to reuse it. It can also be used for protein minipreps as well as pulldown assays using microfuge tubes or mini spin columns. High dynamic binding capacity of purecube 100 ninta agarose.
This resin consists of crosslinked agarose derivatized with nitrilotriacetic acid nta and provides good properties working in native or denaturing conditions. The ni nta resin can be used to purify 6x his tagged proteins under native and denaturing conditions. 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. They are not to be used for human diagnostic or drug purposes. The ni nta resin uses nitrilotriacetic acid nta, a tetradenate chelating ligand, in a highly cross linked 6% agarose matrix.
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