The qia express ninta protein purification system, including the ninta magnetic agarose beads see figure micrograph of ninta magnetic agarose beads is based on the remarkable selectivity of patented ninta nickelnitrilotriacetic acid resin for proteins containing an affinity tag of six or more histidine residues the his tag. The ni nta resin can be used to purify 6x his tagged proteins under native and denaturing conditions. Qiagen ninta agarose, 25ml, 45 to 165m bead, up to 50mgml binding capacity, cell. Synthesis of 53,4bisbenzyloxyphenethylcarbamoylbenzene1,3benzylntalysine the diacid 2 0. Ninta agarose 100 ml from qiagen sample to insight. Ac501 purification histagged proteins nickel nta agarose. Due to the high affinity, ninta magnetic beads can be used for capturing 6x histagged proteins. Jul 28, 2003 all chemicals are purchased from merck except for nintaagarose qiagen, glutathioneagarose sigma and coomassie brilliant blue g 250 serva. Ninta magnetic beads have nitrilotriacetic acid nta groups with charged nickel covalently. Beofore, the protocol is more or less the same as the qiagen provided except that i have incubated the beads with 1% bsa for 1 hr before i added to lysate. Place 50ul beads 100ul suspension of ninta agarose beads in 1. It can also be used for protein minipreps as well as pulldown assays using microfuge tubes or mini spin columns. Ninta magnetic beads have nitrilotriacetic acid nta groups with charged nickel covalently bound to the surface dextran of the beads. Proteins bound to the resin may be eluted with either low.
Automated highthroughput purification of 6xhistagged. High dynamic binding capacity of purecube 100 ni nta agarose. Ni nta agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a his tag. Ninta can then be coupled to agarose resin or magnetic beads for imac immobilized metal chelate affinity chromatography. Gbiosciences ninta magnetic beads are 3m beads designed for the rapid purification of x histagged proteins. Am i right to assume that if the agarose is still light blue, that i just need to wash my resin with 0. Do you have a protocol for the purification of 6xhistagged proteins using. 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. Reusable resin contains 10 mg protein aml of beaded agarose matrix. Ninta resin is guaranteed stable for 6 months when properly stored.
Histagged protein purification, protein and peptide purification, protein sample preparation and protein purification, proteins, expression, isolation and analysis. The tetradentate nta linker generally results in tighter protein binding and reduced metal leaching, compared to the tridentate iminodiacetic idaversion. 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. Affinity histag purification procedure for use nickel nta magnetic agarose beads 5% description nickel nta magnetic agarose beads are products that allow rapid and easy smallscale. Qiagen ni nta agarose, 100ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start material, nickelcharged resin, 2. The polyhistidine tag is the most popular affinity tag and typically consists of six consecutive histidine residues 6xhis. However, since i have seen in the forum that the bsa in fact has no use in blocking the nonspecific binding in the ni nta agarose beads, so i stop using it. Manual purification of 6xhistagged proteins from e. Ni nta magnetic agarose beads are used for smallscale affinity purification as well as highthroughput screening of recombinant histagged proteins. Ninta agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a his tag. Ninta agarose is a nickelcharged affinity resin that can be used to purify recombinant proteins containing a polyhistidine 6xhis sequence. The ni nta resin uses nitrilotriacetic acid nta, a tetradenate chelating ligand, in a highly cross linked 6% agarose matrix.
Nta agarose enables efficient immobilizedmetal affinity chromatography imac using gravityflow chromatography. This allows onestep protein purification under either native or denaturing. This product is not intended for the diagnosis, prevention, or treatment of a disease. Material required the imidazole concentrations of elution buffers 1 and 2 must be optimized for each protein. This resin consists of crosslinked agarose derivatized with nitrilotriacetic acid nta and provides good properties working in native or denaturing conditions. The ni nta purification system is a complete system that includes purification buffers and resin for purifying proteins under native, denaturing, or hybrid conditions. The protein 50 kda dimer is bound to ni nta matrix and then. The protocol describes two cellfree approaches to achieve a selective label scheme. Histidine residues in the his tag bind to the vacant positions in the coordination sphere of the.
Protein purification products cube biotech cube biotech. This video provides generic protocol to purify 6xhistagged protein by nickel nitrilotriacetic ninta sepharose. Check that the resin is contained in the narrow part of the column body before. The nickel nitrilotriacetic acid nta is a qiagenpatented resin which offers affinity purification of 6xhistagged proteins expressed as recombinant proteins from sources like e. Let the resin settle by gravity and gently aspirate the supernatant. Ninta matrices should be stored in 30% ethanol to inhibit microbial growth.
Sonicate or homogenize on ice to lyse cells 6 times for 10 s each time with 5 s pauses between. The ninta resin is precharged and able to bind up to 50 mg of recombinant protein per 1 ml of resin. We are facing purification problem with a histag cloned gene. Once expressed proteins are extracted from cell lysates or membranes, affinity chromatography is an elegant way to purify these to. This product is not intended for the diagnosis, prevention. Add 6 ml of sterile distilled water and resuspend resin.
Nickel columns are used for immobilized metal affinity chromatography imac for the purification of recombinant proteins with a polyhistidine tag on either terminus. High quality ninta products 80 mgml protein cube biotech. Ni1affinitychromatography usestheabilityofhistobindnickel. Qiagen ninta agarose, 100ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate. The purification of histagged proteins consists of 4 stages. Ninta purification system thermo fisher scientific. The protocols given below are for use with ninta superflow columns 1. Ninta agarose and purification columns have the following specifications.
Protein a agarose binds to the fc region of igg from a variety of species. Cgi114 protein concentrations were measured by absorbance at 280 nm mw 26,275. The size of the used agarose resin beads or magnetic beads influences the flow rated and the protein yield. Qiagen ninta agarose, 100ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start. Ni nta can then be coupled to agarose resin or magnetic beads for imac immobilized metal chelate affinity chromatography. Ninta magnetic agarose beads are magnetic particles coated with ninta. The matrix can be stored for up to one week in any of the denaturing buffers. This technology allows onestep purification of almost any. Can probond or ninta beads be used for largescale preparations. Nta chemistry minimizes metal leaching during purification and is compatible with up to 10 mm. Ninta hisbind resin is used for rapid onestep purification of proteins containing a histag sequence by metal chelation chromatography. Protocol for histag based ninta protein purification. All samples were run in duplicate, and the beads were processed using the thermo scientific protocol with buffers recommended by the manufacturers. Nickelnta magnetic agarose beads consist of a ferrimagnetic core that is coated with 6 % crosslinked agarose.
Ni nta column preparation resuspend ni nta agarose slurry in a bottle container. The polyhistidine tag is the most popular affinity 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 proteins using a syringe, peristaltic pump, or liquid chromatography system such as the aktadesign or fplc system. High dynamic binding capacity of purecube 100 ninta agarose. Agarose beads for immunoprecipitation and antibody. 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. Ninta column preparation resuspend ninta agarose slurry in a bottle container. Ninta agarose resin for his tag protein purification. Proteins bound to the resin can be eluted with low ph buffer or competition with imid azole or histidine. This resin can recover histagged proteins from a variety of expression systems such as baculovirus, yeast, mammalian and bacterial cells. Am i right to assume that if the agarose is still light blue, that i just need to. Ninta magnetic agarose beads under native conditions 114 buffer for factor xa protease digestion and removal of factor xa protease with xa removal resin 115 qiaexpress pqe vectors.
The ninta agarose is intended for molecular biology applications. Mar 27, 2007 the ni nta agarose resin is supplied as 50% slurry with ethanol. Ninta magnetic agarose beads are used for smallscale affinity purification as well as highthroughput screening of recombinant histagged proteins. His tag purification purification protocol theoryandintroduction. Gfp was spiked into li lysates and purified on a 1 ml purecube cartridge filled with purecube 100 ninta agarose at flow. Small scale histag purification under nature conditions.
Hi, so in my protocol it says that regeneration should be performed if the ni nta agarose turns from light blue to brownishgray. 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. Ninta hisbind resins store ninta hisbind resin and ninta hisbind superflow at 4c. Highlyefficient purification of native polyhistidine. Can be used to purify classes, subclasses, and fragments of immunoglobulins as. Can be used to purify classes, subclasses, and fragments of immunoglobulins as well as for isolation of immune complexes. Perform routine affinity purification of histagged fusion proteins with thermo scientific hispur ninta resin, a highcapacity, highperformance nickelimac resin.
Dear all, i am using ninta agarose qiagen for purification of histagged proteins by gravityflow chromatography. Proteins bound to the resin may be eluted with either low ph buffer or by competition with imidazole or histidine. 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. Ni nta agarose is a nickelcharged affinity resin that can be used to purify recombinant proteins containing a polyhistidine 6xhis sequence. Pressure, manualautomated processing, large scale, sepharose cl6b matrix, 100g to 100mg yield, 6xhis. This is a purification method to obtain functional his tagged protein. Hispur ninta beads provided higher yield and purity than qiagen ninta agarose magnetic beads for purification of 6xhis. Purification of recombinant proteins using the qia express system does not depend on the 3dimensional structure of the protein or 6xhis tag. How imidazole can be washed off the ninta agarose beads. Nickel nta agarose beads are provided readytouse for rapid purification of histagged proteins under native or denaturing conditions. Protocol ninta magnetic agarose beads handbook 122001 21 4. Ni nta superflow 500 ml 500 ml nickelcharged resin max. Hi, so in my protocol it says that regeneration should be performed if the ninta agarose turns from light blue to brownishgray. For more detailed information see the manufacturers handbook provided with the purification matrix.
Ni nta magnetic agarose beads under native conditions 114 buffer for factor xa protease digestion and removal of factor xa protease with xa removal resin 115 qiaexpress pqe vectors. Qiagen ni nta agarose, 25ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start material, nickelcharged resin, 2. Protein purification with the ninta protein purification system. Qiagen ninta agarose, 25ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start.
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