| Product Description | Aladdin's UltraBio™ IDA-Ni Magnetic Agarose Beads for His-Tag Protein Purification are made by covalently coupling high-quality Iminodiacetic Acid (IDA) to agarose beads and then chelating divalent nickel ions (Ni2+) through the three binding sites of IDA. This product can specifically bind to proteins containing His-tags 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. This product is tolerant to 6M guanidine hydrochloride or 8M urea.The 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 the 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]. Generally, His-tagged protein purification media (nickel column) is used to purify His-tagged proteins, but for applications such as a small amount of purification and immunoprecipitation of His-tagged fusion proteins or protein complexes, Anti-His magnetic beads are simpler and more convenient to use.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) chelating agents. IDA, NTA, and TED have 3, 4, and 5 coordination sites, respectively, for binding nickel ions, which means that a nickel ion has 3, 2, and 1 available binding sites, respectively, for histidine-tagged proteins. Therefore, IDA has the highest binding capacity for His-tagged proteins, but with relatively lower binding specificity, and 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 with His-tagged fusion proteins. With a magnetic stand, it can be conveniently used for protein purification and immunoassays. Please refer to Figure 1 for the workflow of this product.Figure 1. The workflow of UltraBio™ IDA-Ni Magnetic Agarose Beads for His-Tag Protein Purification High specificity and high binding capacity
Precautions： Repeated freeze-thaw of this product up to 3 cycles does not cause obvious change of its performance. However, it is still recommended to aliquot this product to avoid repeated freeze-thaw. For frequent use, we recommend storing at 4℃.This product should be maintained at a pH of 6-8. Do not centrifuge, dry or freeze 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 and 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 explored.Resuspend the magnetic agarose beads thoroughly in solution by gentle pipetting prior to use. Do not vortex.When purification, it is recommended to set up both positive and negative controls appropriately.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 the aspiration of magnetic beads.Aggregation of magnetic agarose beads can be effectively prevented by adding 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 and NP-40 Lysis Buffer are fully applicable. For the 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 described for the purification of His-tagged recombinant proteins expressed in Escherichia coli. Typically, each 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 the target protein, 2ml of magnetic agarose bead slurry is required. This volume can be scaled up or down according to the specific needs of the experiment.1. Preparation of buffersBufferComponentsBinding/Wash Buffer10mM Tris, 500mM NaCl, pH7.4Elution Buffer10mM Tris, 500mM NaCl, 500mM Imidazole, pH7.4Note 1: It is recommended to filter the solutions with 0.22μm or 0.45μm filter membranes before use to improve the purification efficiency of His-tagged proteins.Note 2: The buffers recommended above are suitable for the purification of most His-tagged proteins. For special protein samples, appropriate optimization may be required or FAQ in this manual can be referred to.2. Preparation of protein samplesa. Centrifuge the Escherichia coli culture at 4℃ for 20 minutes at 4,000×g or 1 minute at 15,000×g to collect cells. Discard the supernatant and collect the pellet. Proceed to bacterial lysis or store the pellet at -20℃ or -80℃ for later use. Thaw the frozen cells on ice for 15 minutes before use.b. Resuspend the bacterial pellet thoroughly in Binding/Wash Buffer. If necessary, add an appropriate amount of protease inhibitors, such as the Protease Inhibitor Cocktail for His-tagged Protein Purification (100X) ( Super Nuclease (≥99%) ( Digital Rocking Platform .b. After incubation, place the mixture on a magnetic rack for 10 seconds, remove and discard the supernatant. c. Note: A small amount of supernatant can be reserved in a clean microcentrifuge tube for examination of the binding efficiency.d. Wash beads with 2ml of Binding/Wash Buffer by gentle pipetting to resuspend the IDA-Ni beads completely. Place the tube on a magnetic rack for 10 seconds, remove and discard the supernatant. Repeat this step thrice.5. Elutiona. Based on the concentration 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 IDA-Ni beads and incubate for 5 minutes. Place the tube on a magnetic rack for 10 seconds and collect the supernatant containing the purified His-tagged proteins into a new centrifuge tube.b. If needed, perform a second elution to improve the yield.6. Regeneration of IDA-Ni beadsa. After protein elution, add 2ml of Elution Buffer to the IDA-Ni beads and resuspend the beads by inverting the tube several times. Vortex for 10 seconds, place the tube on a magnetic rack for 10 seconds, remove and discard the supernatant. Repeat this step thrice.b. Add 2ml of 20% ethanol and resuspend the beads by inverting the tube several times. Vortex for 10 seconds, place the tube on a magnetic rack for 10 seconds, remove and discard the supernatant. Repeat this step thrice.c. Store the magnetic beads in an equal volume of 20% ethanol to the initial suspension. Store at 2-30℃ (for long-term storage, store at 2-8℃). The beads can be reused 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 target protein has His-tag by Western blot or dot blot analyses.Very low protein expression level.1. Use more cells or tissues for lysis.2. Optimize expression conditions to increase the protein expression level.Washes are too stringent.Reduce the time and number of washes.Incubation times are 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.If Western blot detection is used:1. Check the specificity of primary and secondary antibodies using proper controls.2. Check the protein transfer efficiency by using a 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 sample.Try a gradient pH elution or a gradient imidazole elution. Insufficient washing of IDA-Ni beads.1. Increase the number of washes.2. Prolong duration of each wash to at least 15 minutes.3. Add 5-20mM imidazole in the Wash Buffer.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.
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