{"product_id":"nhs-activated-beads-ua100039","title":"NHS-Activated Beads","description":"\u003ch4\u003eProduct Specification\u003c\/h4\u003e\u003cdiv class=\"responsive-table product-detail-table details-table\"\u003e\n\u003cbr\u003e\u003ctable style=\"width: 100%; height: auto;\"\u003e\u003ctbody\u003e\u003ctr\u003e\n\u003ctd style=\"width: 22%;\"\u003e\u003cstrong\u003ePhysical Appearance\u003c\/strong\u003e\u003c\/td\u003e\n\u003ctd style=\"width: 78%;\"\u003eLiquid\u003c\/td\u003e\n\u003c\/tr\u003e\u003c\/tbody\u003e\u003c\/table\u003e\n\u003c\/div\u003e\u003ch4\u003eBackground\u003c\/h4\u003e\u003cdiv\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eNHS-Activated Beads are pre-activated agarose microspheres that can be directly used for coupling with amino-containing proteins or peptides. The pre-activated medium can be prepared into specialized affinity media as needed, enabling rapid and efficient one-step purification of target substances from complex systems. NHS-Activated Beads exhibit excellent pressure resistance and stable performance after protein coupling, making them suitable for large-scale industrial purification.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003ctable class=\"table table-bordered\"\u003e\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:3.0000pt\"\u003eH\u003cbr\u003e\u003c\/span\u003e\u003cb\u003e\u003cspan style=\"color:rgb(0,0,0);font-weight:bold;font-size:10.0000pt\"\u003e  \u003cfont\u003eItem\u003c\/font\u003e\u003c\/span\u003e\u003c\/b\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd\u003e\n\u003cp\u003e\u003cb\u003e\u003cspan style=\"color:rgb(0,0,0);font-weight:bold;font-size:10.0000pt\"\u003e \u003c\/span\u003e\u003c\/b\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cb\u003e\u003cspan style=\"color:rgb(0,0,0);font-weight:bold;font-size:10.0000pt\"\u003e\u003cfont\u003ePerformance\u003c\/font\u003e\u003c\/span\u003e\u003c\/b\u003e\u003c\/p\u003e\n\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e  \u003cfont\u003eMatrix\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e\u003cfont\u003eHighly cross-linked\u003c\/font\u003e 4% agarose microspheres\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e  \u003cfont\u003eCoupling capacity\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e\u0026gt;10 mg IgG\/ml medium\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e  \u003cfont\u003eParticle size range\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e45-165μm\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e  \u003cfont\u003eMaximum pressure\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e0.3 MPa, 3 bar\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e  \u003cfont\u003eStorage buffer\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e100% isopropanol\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e  \u003cfont\u003eStorage temperature\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd\u003e\u003cp\u003e\u003cspan style=\"color:rgb(0,0,0);font-size:10.0000pt\"\u003e-20℃\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\u003c\/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c\/p\u003e\n\u003c\/div\u003e\u003ch4\u003eProtocol\u003c\/h4\u003e\u003cdiv\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e\u003cstrong\u003e2.1 Preparation of Buffer Solutions\u003c\/strong\u003e\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eIt is recommended to filter all water and buffer solutions through a 0.22 μm or 0.45 μm membrane before use.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eWashing solution: 1 mM HCl\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eCoupling solution: 0.2 M NaHCO3, 0.5 M NaCl, pH 8.0\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eBlocking solution: 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3 or 0.1 M Tris, pH 8.5\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eWashing solution 1: 0.1 M acetate-sodium acetate, 0.5 M NaCl, pH 3.0\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eWashing solution 2: 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003ePreservation solution: 1X PBS containing 20% ethanol\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eNote: 1. The coupling solution can be selected from carbonate, phosphate, or other buffer systems without amino groups. Adding a certain concentration of salt ions to the buffer system can reduce non-specific adsorption.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e2. \u003cfont\u003eWhen the coupling sample is an antibody, 1X PBS with 0.02% NaN3 or 1X PBS with 0.02% Proclin 300 can be used as the preservation solution.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e \u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e\u003cstrong\u003e2.2 Sample Preparation\u003c\/strong\u003e\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eDissolve or dialyze the sample in the coupling solution, with a concentration of approximately 5-10 mg\/ml.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e \u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e\u003cstrong\u003e2.3 Sample Coupling\u003c\/strong\u003e\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eThe following example uses antibody coupling to purify antigens, introducing the coupling and subsequent purification steps.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e1) Take an appropriate amount of NHS-Activated Beads, wash three times with the washing solution by suction filtration, and wash once with the coupling solution. Note: Do not dry the beads too much to avoid clumping. If clumping occurs, disperse by shaking or pipetting. Pre-cooled solutions can be used for rapid washing to reduce hydrolysis of pre-activated media.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2) Add the dissolved sample to the washed NHS-Activated Beads, with a volume ratio of NHS-Activated Beads to sample solution of approximately 1:1-2 (V\/V).\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e3) Shake and react at 28°C for 2-4 hours or at 4°C overnight. Note: Ensure the beads are suspended; otherwise, coupling efficiency will be greatly affected.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e4) After the reaction, collect the coupled sample for efficiency testing. Wash the beads with deionized water, add 2 times the column volume of blocking solution, and shake at 28°C for 1 hour.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eNote: After coupling, the supernatant protein concentration cannot be measured by UV absorption. It is recommended to use electrophoresis or BCA quantification to test coupling efficiency.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e5) Remove the reaction system, drain the blocking solution, wash the beads with 3 times the column volume of deionized water, and repeat washing with washing solution 1, deionized water, washing solution 2, and deionized water twice. Then store in an equal volume of preservation solution at 2-8°C.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e \u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e\u003cstrong\u003e2.4 Column Packing\u003c\/strong\u003e\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2.4.1 Gravity Column Packing\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e1) Take a gravity column of appropriate specification, insert the lower frit, add an appropriate amount of pure water to rinse the column tube and frit, and close the lower outlet.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2) Mix the coupled beads evenly, pipette an appropriate amount of slurry into the gravity column, and open the lower outlet to drain the preservation solution.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e3) Add an appropriate amount of pure water to rinse the medium. After the liquid in the column tube drains by gravity, close the lower outlet.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e4) Insert the rinsed upper frit, ensuring no gaps between the frit and the beads and keeping it level.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e5) The packed gravity column can directly add equilibration buffer for balancing.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2.4.2 Medium-Pressure Column Packing\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eCoupled beads can also be used for large-volume sample purification, involving various medium-pressure chromatography column packing methods. Below is a method for packing chromatography columns.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eBefore packing, calculate the column bottom area based on the column diameter, and calculate the required medium volume based on the desired packing height using the following formula:\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cimg class=\"img-fluid\" src=\"data:image\/png;base64,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\"\u003e\u003cbr\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eV: Required medium volume (ml)\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e1.15: Compression coefficient\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003er: Column tube radius (cm)\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eh: Packing height (cm)\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eNote: The volume of the suspension taken should be twice the medium volume, as the medium volume only accounts for half of the total suspension volume, with the other half being the preservation solution.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e1) Rinse the column bottom frit and connector with deionized water to ensure no bubbles on the frit. Close the column bottom outlet and leave 1-2 cm of deionized water at the bottom.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2) Suspend the beads and carefully pour the slurry continuously into the column. Pouring the slurry along the column wall with a glass rod can reduce bubble formation.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e3) If using a reservoir, immediately fill the column and reservoir with water. Place the sample distributor on the slurry surface and connect it to the pump to avoid bubble formation in the distributor or inlet tube.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e4) Open the column bottom outlet, start the pump, and run at the set flow rate. Initially, let the buffer flow slowly through the column, then gradually increase to the final flow rate to avoid hydraulic impact on the formed bed and ensure uniform packing. If the recommended pressure or flow rate cannot be achieved, use the maximum flow rate of your pump for good packing results. (Note: Do not exceed 75% of the maximum packing flow rate in subsequent chromatography procedures.) When the bed height stabilizes, run at least 3 times the bed volume of deionized water at the final packing flow rate. Mark the bed height.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e5) Turn off the pump and close the column outlet.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e6) If using a reservoir, remove it and place the distributor in the column.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e7) Push the distributor to the marked bed height. Allow the packing solution to enter the distributor and tighten the distributor connector.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e8) Connect the packed column to the pump or chromatography system and start equilibration. Adjust the distributor if necessary.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e \u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e\u003cstrong\u003e2.5 Sample Purification\u003c\/strong\u003e\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2.5.1 Preparation of Buffer Solutions\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eIt is recommended to filter all water and buffer solutions through a 0.22 μm or 0.45 μm membrane before use.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eEquilibration\/Wash buffer: 0.15 M NaCl, 20 mM Na2HPO4, pH 7.0\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eElution buffer: 0.1 M glycine, pH 3.0\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eNeutralization buffer: 1 M Tris-HCl, pH 8.5\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2.5.2 Incubation Purification\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e1) Based on the sample volume, add an appropriate amount of coupled beads to the column and drain the preservation solution by gravity.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2) Wash the beads with 5 times the medium volume of equilibration buffer and drain by gravity.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e3) Add the sample, seal both ends of the column, and incubate at 4°C with shaking for 2-4 hours or at 37°C for 30 minutes to 2 hours.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e4) After incubation, centrifuge or filter to collect the beads, retaining the supernatant as flow-through for electrophoresis analysis.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e5) Wash the beads with 5 times the medium volume of wash buffer, centrifuge or filter to remove the supernatant, and repeat 3-5 times, changing tubes as needed.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e6) Add 3-5 times the column volume of elution buffer for elution, incubate for 10-15 minutes, and collect the eluate by centrifugation or filtration. Repeat 2-3 times. Neutralize the eluate immediately, typically using 1\/10 volume of neutralization buffer.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2.5.3 Gravity Column Purification\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e1) Equilibrate the packed gravity column with 5 times the column volume of equilibration buffer to condition the beads in the same buffer system as the target protein.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2) Load the sample onto the equilibrated gravity column, collect the flow-through, and repeat loading to increase binding efficiency.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e3) Wash with 10 times the column volume of wash buffer to remove non-specifically adsorbed impurities, collecting the wash fractions.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e4) Elute with 5 times the column volume of elution buffer, collecting fractions. Neutralize the eluate immediately, typically using 1\/10 volume of neutralization buffer.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2.5.4 Medium-Pressure Column Purification\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eAfter packing, medium-pressure columns can be used with various conventional medium\/low-pressure chromatography systems.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e1) Fill the pump tubing with deionized water. Remove the upper cap, connect the column to the chromatography system, open the lower outlet, attach the pre-packed column, and tighten.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e2) Rinse out the storage buffer with 3-5 times the column volume of deionized water.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e3) Equilibrate the column with at least 5 times the bed volume of equilibration buffer.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e4) Load the sample using the pump or sample loop.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eNote: Increased sample viscosity can cause high backpressure even with small sample volumes. Do not exceed the column's binding capacity. Large sample volumes may also cause high backpressure, making injection difficult.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e5) Wash the column with wash buffer until UV absorption reaches a stable baseline (typically at least 10-15 column volumes).\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e6) Elute with 5-10 times the column volume of elution buffer, collecting the eluate (target protein fractions). Neutralize the eluate immediately, typically using 1\/10 volume of neutralization buffer.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eAfter elution, rinse the beads with 5-10 column volumes of equilibration buffer, then 5-10 column volumes of pure water, and finally 2 column volumes of 20% ethanol. Store at 2-8°C.\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e \u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003e\u003cstrong\u003e2.6 SDS-PAGE Detection\u003c\/strong\u003e\u003c\/font\u003e\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:10.5000pt\"\u003e\u003cfont\u003eAnalyze the samples obtained from purification (including flow-through, wash, and elution fractions) and the original sample using SDS-PAGE to evaluate purification efficiency.\u003c\/font\u003e\u003c\/span\u003e\u003cbr\u003e\u003c\/p\u003e\n\u003c\/div\u003e","brand":"UA BIOSCIENCE","offers":[{"title":"1ml","offer_id":43090002509899,"sku":"UA100039-1ml","price":83.33,"currency_code":"USD","in_stock":true},{"title":"5ml","offer_id":43090002542667,"sku":"UA100039-5ml","price":291.67,"currency_code":"USD","in_stock":true}],"thumbnail_url":"\/\/cdn.shopify.com\/s\/files\/1\/0590\/8375\/1499\/files\/AntBioImage_c733b07d-78c3-4ec2-afcb-84e5c0c93026.png?v=1788112949","url":"https:\/\/www.antbioinc.com\/products\/nhs-activated-beads-ua100039","provider":"AntBio","version":"1.0","type":"link"}