| Product Description | Aladdin's UltraBio™ NTA-Ni Magnetic Agarose Beads for His-Tag Protein Purification are generated by covalently coupling high-quality Nitrilotriacetic acid (NTA) to agarose beads and then chelating divalent nickel ions (Ni2+) through the four binding sites of NTA. This product can specifically bind to His-tag fusion proteins in cell lysates, serum, ascites fluid, and other samples from animals, plants, or microorganisms, and thus can be used for purification, immunoprecipitation (IP), or co-immunoprecipitation (Co-IP) of proteins or protein complexes with His-tags. It is tolerant to 6M guanidine hydrochloride or 8M urea.His-tag is a peptide consisting of 6 consecutive histidine residues (HHHHHH), typically fused to the N or C terminus of a recombinant protein. It is small in size (0.84kDa only) and usually does not interact with the target protein, neither does it affect the function of the target protein in most cases, nor have any impacts on downstream applications of the protein. When fused with His-tag, the expression, localization and function of the target protein can be analyzed using His antibody and Anti-His magnetic beads, and the target protein can also be purified, immunoprecipitated or co-immunoprecipitated. Purification of His-tagged proteins are simple, with mild purification conditions. The His-tag can be expressed in a variety of protein expression systems and can be used together with other tags to generate dual-affinity tag proteins. The N-terminal His-tag is compatible with the bacterial transcription and translation mechanism, which is beneficial for protein expression. Based on the above advantages, the His-tag has been widely used for protein expression, purification, identification, interaction and function studies [1]. While conventional His-tag protein purification resin (nickel column) is generally used for purifying His-tagged proteins, NTA-Ni Magnetic Agarose Beads offer a simpler and more convenient option for purifying low amounts of His-tagged proteins or protein complexes, and for immunoprecipitation as well.This product is developed based on the immobilized metal affinity chromatography (IMAC). A nickel ion has six coordination sites, and the bridges connecting it to agarose or magnetic agarose beads are typically IDA (Iminodiacetic acid), NTA (Nitrilotriacetic acid), or TED (Tris(carboxymethyl)ethylenediamine), which are chelating agents. IDA, NTA, and TED have 3, 4, and 5 coordination sites, respectively, for binding to nickel ions, which means that they have 3, 2, and 1 available binding sites, respectively, for His-tagged proteins. Therefore, IDA has the strongest binding affinity and highest binding capacity for His-tagged proteins, but with relatively weaker specificity. The His-tagged proteins can be efficiently eluted with a low concentration of imidazole, while TED is the opposite. NTA's interaction with His-tagged proteins falls in between the other two [2].This product specifically binds His-tag fusion proteins. With a magnetic rack, it can be conveniently used for protein purification and immunoassays. Please refer to Figure 1 for the workflow of immunoprecipitation using this product.Figure 1. The workflow of Aladdin's UltraBio™ NTA-Ni Magnetic Agarose Beads for His-Tag Protein Purification .High specificity and high binding capacity
Precautions： This product can maintain its activity after three freeze-thaw cycles. However, it is still recommended to minimize the number of freeze-thaw cycles by properly aliquoting the beads. For frequent use, storage at 4℃ is recommended.This product should be maintained at pH 6-8. Do not centrifuge or dry the magnetic beads. Long time exposure of the beads to magnetic field will cause beads to agglomerate.During the use of this product, the concentration of buffer reagents, such as Tris, HEPES, and MOPS, should not exceed 100mM. Avoid using chelating agents such as EDTA or EGTA, as well as reducing agents such as Glycine, DTT, and DTE. The concentration of Triton, Tween, and NP-40 should not exceed 2%. The concentration of deoxycholic acid sodium salt and CHAPS should not exceed 1%. The concentration of histidine and calcium ions should not exceed 20mM and 5mM, respectively. The maximum concentration of hydrochloric guanidine and urea is 6M and 8M, respectively. The concentration of sodium ions and magnesium ions can be up to 2M, while the concentration of glycerol can be up to 50%. Its compatibility with other unspecified reagents has not been tested and should be experimentally exploredThis product should be fully resuspended in solution by gentle pipetting prior to use. Do not vortex to avoid the denaturation of the antibody.Both positive and negative controls should be set up appropriately for protein purification or immunoprecipitation.Protein samples should be purified as soon as possible after collection and should always be placed at 4℃ or on ice to minimize protein degradation or denaturation. Protein degradation can also be inhibited by adding appropriate protease inhibitors, such as Protease Inhibitor Cocktail for General Use , Protease and Phosphatase Inhibitor Cocktail for General Use (MS-safe, 50X) , Protease Inhibitor Cocktail for Mammalian Cell and Tissue Extracts , Protease and Phosphatase Inhibitor Cocktail for Mammalian Cell and Tissue Extracts , etc.If using a vacuum pump to aspirate the supernatant, the strength of the vacuum pump should be controlled properly to avoid disturbing the magnetic beads.Aggregation of Magnetic Agarose Beads can be effectively prevented by including 0.1% non-ionic detergents, such as Triton X-100, Tween-20, and NP-40, which will not affect the binding efficiency of the beads.High concentrations of DTT, mercaptoethanol, and guanidine hydrochloride may have a certain impact on the binding of this product to tagged proteins, but Cell lysis buffer for Western and IP , RIPA Lysis Buffer or NP-40 Lysis Buffer are fully applicable. For selection guide of different lysis buffers from , please refer to our website at http://www.aladdin-e.com/support/lysis-buffer.htm.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： This protocol is provided for the purification of His-tagged recombinant proteins expressed in E. coli. Typically, 500ml of bacterial culture yields 2-4g wet weight of cells, and the target protein yield is expected to be 10-20mg. For the purification of target protein, 2-4ml of magnetic bead suspension is required. This volume can be scaled up or down as required.1. Buffer preparationBufferComponentsBinding/Wash Buffer10mM Tris, 500mM NaCl, 20mM Imidazole, pH7.4Elution Buffer10mM Tris, 500mM NaCl, 20mM Imidazole, pH7.4Note 1: It is recommended to filter the water and buffers used with a 0.22μm or 0.45μm filter membrane before use to reduce impurities and improve the purification efficiency of His-tagged proteins.Note 2: The recommended buffers are suitable for the purification of most His-tagged proteins. For special samples, optimization may be required or FAQ in this manual can be referred to.2. Protein sample preparationa. Collect the Escherichia coli culture into a centrifuge tube, centrifuge at 4,000×g for 20 minutes at 4℃ or 15,000×g for 1 minute. Discard supernatant. Proceed to bacteria lysis immediately, or freeze the pellet at -20℃ or -80℃ for later use. The frozen bacteria should be thawed on ice for 15 minutes prior to bacteria lysis.b. Resuspend the pellet in Binding/Wash Buffer. If necessary, add an appropriate amount of protease inhibitor cocktail, such as the Protease Inhibitor Cocktail for His-tagged Protein Purification (100X) ( Super Nuclease (≥99%) ( NTA-Ni Magnetic Agarose Beads (NTA-Ni beads)a. Gently resuspend the NTA-Ni beads by pipetting and transfer an appropriate amount to a new centrifuge tube ( Digital See-Saw Rocking Shaker (, E6673).b. After incubation, place the tube on a magnetic rack for 10 seconds and remove the supernatant. Note: A portion of the supernatant can be collected to test the binding effect of the target protein.c. Add 2ml of Binding/Wash Buffer and gently resuspend the magnetic beads by pipetting. Place the tube on a magnetic rack for 10 seconds and then remove the supernatant. Repeat the wash three times.5. Elutiona. Based on the abundance of the target protein and the requirements of downstream applications, add 0.2-1ml of Elution Buffer. Gently invert the centrifuge tube a few times to suspend the beads and incubate for 5 minutes. Perform magnetic separation and collect the eluate into a new centrifuge tube. The eluate contains the purified His-tagged proteins.b. If needed, repeat the elution one more time to increase the protein yield.6. Washing and regeneration of the NTA-Ni beadsa. Resuspend the magnetic beads with 2ml of Elution Buffer. Vortex for 10 seconds, place the tube on a magnetic stand for 10 seconds, and remove the supernatant. Repeat this step 3 times.b. Add 2ml of 20% ethanol to the centrifuge tube. Invert the tube several times to resuspend the magnetic beads. Vortex for 10 seconds, perform magnetic separation, and remove the supernatant. Repeat this step 3 times.c. Store the magnetic beads in 20% ethanol of an equal volume to the initial suspension volume. Store at 2-30℃ for later use, or at 2-8℃ for long-term storage. The regenerated beads can be used for the purification of the same protein.ProblemsPossible CausesSolutionsVery few or no His-tagged protein exists in the eluate.Protein is not completely eluted.1. Increase the concentration of imidazole in Elution Buffer.2. Extend the elution time.No target protein expressed.Make sure the protein of interest contains the His-tag by Western blot or dot blot analyses.Very low protein expression level.1. Use larger volume of cell lysate.2. Optimize expression conditions to increase the protein expression level.Washes are too stringent.Reduce the wash time and number of washes.Incubation time is inadequate.Increase the incubation time.Interfering substance is present in sample.Lysate contains high concentration of DTT, 2-mercaptoethanol, or other reducing agents that may interfere with the binding of His-tagged proteins to the IDA-Ni beads.Low sensitivity of the detection system.For Western blot analysis:1. Check the specificity and reactivity of primary and secondary antibodies using proper controls.2. Check the protein transfer efficiency by using prestained protein marker or staining the membrane with Ponceau S.3. Use fresh ECL western substrate or try a different ECL substrate with higher sensitivity.The purity of the target protein is low.There are other polyhistidine-rich proteins in sampleTry a pH or imidazole gradient elution. Insufficient washing of beads after protein binding.1. Increase the number of washes.2. Prolong duration of the washes, incubating each wash for at least 15 minutes.3. Add 5-20mM imidazole in the wash solutions.4. Centrifuge at lower speed to avoid nonspecific trapping of denatured proteins.The binding capacity of the magnetic beads has declined.Aggregation of protein or other substances on the beads.Wash the beads with NaOHThe beads have been reused too many times.Use new beads.References:1. Bromberg R, Cai K, Guo Y, Plymire D, Emde T, et al. Front Mol Biosci. 2022. 9:912072.2. Zeng K, Sun EJ, Liu ZW, Guo J, Yuan C, et al. RSC Adv. 2020. 10(19):11524-11534.
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