| Product Description | Aladdin's UltraBio™ Concanavalin A Magnetic Beads are based on superparamagnetic nanoscale beads covalently coupled with high-quality Concanavalin A (ConA) that specifically binds to glycoproteins, glycolipids, polysaccharides and other glycosylated molecules rapidly and efficiently. This product is mainly used for separating cells, for isolating glycoproteins and other glycosylated molecules from cell or tissue lysates or serum, and for Cleavage Under Targets & Release Using Nuclease (CUT&RUN) in chromatin profiling.ConA is a Ca2+/Mn2+-dependent mannose/glucose-binding lectin isolated from jack beans (Canavalia ensiformis). It is a T-cell mitogen that activates the immune system, recruits lymphocytes and induces cytokine production. In addition to its mitogenic activity, ConA can induce programmed cell death through specific molecular mechanisms. ConA has also been reported to activate NFAT (Nuclear factor of activated T cells), a family of transcription factors that play an important role in the development and function of the immune system. ConA exists as a tetramer when the pH is between 5.8 and 7.0, dissociates into a dimer at pH 6.5 or lower, and can form larger aggregates at pH higher than 7.0 [1].UltraBio™ ConA Magnetic Beads are easy to use to isolate or fix cells and minimize cell loss during subsequent washing, and can also be used to collect and fix cell nuclei [2-4]. Please refer to Figure 1 for the procedure of glycoprotein purification using ConA magnetic beads. The work flow for isolation or fixation of cells or nuclei is similar.Figure 1. The work flow of UltraBio™ Concanavalin A Magnetic Beads for purification of glycoproteins.High Specificity
Precautions： This product should be maintained at pH 6-8. Do not centrifuge, dry or freeze the magnetic beads. Long time exposure of the beads to magnetic field will causes beads to agglomerate.Resuspend Mag™ Anti-HA Magnetic Beads evenly in solution by gentle pipetting prior to use. Do not vortex to avoid the denaturation of ConA.When performing precipitation or purification, it is recommended to set up both positive and negative controls appropriately.The type and size of the molecule to be isolated will affect the binding efficiency. It is recommended to determine the amount of beads for each specific application by dilution method, and the amount of beads is recommended to be 2-3 times the molar amount of the molecule to be isolated to ensure sufficient binding.ConA requires the presence of Ca2+ and Mn2+ ions to be active, so reagents containing EDTA or other metal ion chelators should not be used during the experiment.The glycoprotein 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. However, these protease inhibitor cocktails should not contain EDTA or other metal ion chelators.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.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： The following protocol is provided for glycoprotein purification or cell isolation. CUT&RUN experiments require special methods. 1. Buffer Preparation.BufferComponentsBinding Buffer20mM Hepes (pH7.4), 150mM NaCl (optional), 1mM MgCl2, 1mM MnCl2, 1mM CaCl2Wash Buffer20mM Hepes (pH7.4), 150mM NaCl (optional), 1mM MgCl2, 1mM MnCl2, 1mM CaCl2, 0.1% Tween-20Elution Buffer5mM Tris (pH8.0), 150mM NaCl, 1M GlucoseNote 1: We recommend using Hepes buffer, instead of PBS buffer that may cause precipitation. Equilibrate all buffers to room temperature prior to use. The addition of 150 mM NaCl to Binding Buffer and Wash Buffer is optional, which is not recommended for CUT&RUN and other experiments where cell viability is not important, and recommended if the isolated cells are subsequently used for cell culture or cell function studies. The efficiency of cell binding and precipitation is higher without the addition of 150 mM NaCl, and the addition of 150 mM NaCl better maintains the osmolarity of cells.Note 2: Elution Buffer requires certain optimization depending on the type of glycoprotein or the binding capability of glycoprotein to ConA magnetic beads. Note 3: Since cells can bind tightly to ConA magnetic beads, this Elution Buffer is not recommended to be used for cell elution.2. Sample Preparation (Using mammalian cells for example).a. For adherent cells: Remove the culture medium, wash twice with 20mM Hepes (pH7.5) containing 150mM NaCl. Digest the cells by adding 100-200μl Trypsin Solution per well of a 6-well plate. After 1-2 min digestion, wash and resuspend the cells with 20mM Hepes (pH7.5) containing 150mM NaCl, and then centrifuge at 400-500×g for 5 min at room temperature. Remove the supernatant and proceed to step c.b. For suspension cells: Centrifuge at 400-500×g for 5 min at room temperature to collect the cells, remove the supernatant. Wash and resuspend the cells with 20mM Hepes (pH7.5) containing 150mM NaCl, and then centrifuge at 400-500×g for 5 min at room temperature. Remove the supernatant and proceed to step c. c. Cell Resuspension: Add an appropriate amount of Binding Buffer and fully resuspend cells gently. Perform cell counting under a microscope. The number of cells per sample should be 10,000-100,000 in 500μl. Calculate the volume of cell suspension required for each sample and dilute the cell suspension with Binding Buffer.d. For cell or tissue lysate or serum samples: Dilute with Binding Buffer and adjust the dilution factor based on the experimental result.3. Preparation of ConA Magnetic Beads.a. Wash: Since the ConA magnetic beads are stored in a special protective solution, they need to be washed twice with 10 times volume of Binding Buffer before adding to samples.(a) Resuspend the ConA beads completely by gently pipetting, take an appropriate amount of ConA beads at a ratio of 10μl of ConA beads suspension per 200-500μl of sample into a clean centrifuge tube and add 10 times the volume of Binding Buffer. (b) Resuspend the ConA beads by gently pipetting, place the tube on a magnetic stand for 10 seconds to collect the beads against the side of the tube. Remove and discard the supernatant. (c) Repeat the wash once. b. Activation: Resuspend the ConA magnetic beads with Binding Buffer according to the initial volume of ConA magnetic beads.Note 1: For multiple samples, take the total required amount of magnetic beads, wash and process them together and then dispense them equally into individual samples.Note 2: The activated beads should be used on the same day as they are prepared.4. Binding of Samples.a. Incubation: Add an appropriate amount of sample and resuspend the magnetic beads by gently pipetting. Place the tube on a rotating mixer and incubate for 10-30 minutes at room temperature or 4-16 hours at 4℃.Note 1: ConA magnetic beads may aggregate during incubation, which is a normal phenomenon and does not affect the result.Note 2: To ensure complete binding of the sample to the beads, the amount of ConA beads can be increased and the incubation time can be extended.b. Magnetic Separation: Place the tube on a magnetic stand for 1 minute to collect the beads against the side of the tube. Remove and discard the supernatant..c. Wash: Add 0.5ml of Wash Buffer to resuspend the beads by gently pipetting, wash for 5 minutes on a rotating mixer. Place the tube on a magnetic stand for 1 minute to collect the beads and remove the supernatant. Repeat the wash 3-4 times.5. Elution of Glycoprotein.a. Add 50-250μl of Elution Buffer to each sample, mix well and incubate at room temperature for 10-30 minutes on a rotating mixer. To increase the elution efficiency, repeat the elution once and combine the eluates, or increase the incubation time.b. Place the tube on a magnetic stand for 10 seconds, and transfer the supernatant into a new centrifuge tube, which should contain the purified glycoprotein.c. For SDS-PAGE electrophoresis or Western blot, mix the eluate with an appropriate amount of 5X SDS-PAGE loading buffer, heat at 95℃ for 5 minutes, place on a magnetic stand for 10 seconds and take the supernatant for subsequent assay. Note: Elution Buffer needs to be optimized for different types of glycoprotein[5]. Alternatively, the PNGase F enzyme can be used to cleave the glycosyl group, thus dissociating the glycoprotein from the magnetic beads. 6. Elution of Cells.Theoretically, the cells can be used without elution from the beads, because the magnetic beads are relatively small and do not affect cell growth and activity. References:1. Dwyer JM, Johnson C. Clin Exp Immunol. 1981.46(2):237-249.2. Hainer SJ, Fazzio TG. Curr Protoc Mol Biol. 2019. 126(1):e85.3. Skene PJ, Henikoff S. Elife. 2017. 16(6):e21856.4. Skene P, Henikoff J, Henikoff S. Nat Protoc. 2018. 13(1006-1019).5. Soper AS, Aird SD. J Chromatogr A. 2007. 1154(1-2):308-18.
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