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