AffiAmino UltraRapid Agarose - PDF Free Download

Table of Contents 1. General information... 3 2. Principle of action... 4 3. Material supplied... 4 4. Additional materials needed... 4 5. General handling... 4 6. Coupling and Binding Protocol... 5 7. Practical notes... 6 8. Disclaimer... 6 9. Ordering information... 7 2

1. General information AffiAmino UltraRapid Agarose consists of super-paramagnetic agarose beads which are functionalized with amino-reactive groups. They can be used for covalent coupling of molecules with primary amino-groups in e.g. proteins such as antibodies, enzymes and aptamers. Subsequently, target proteins can be affinity purified using magnetic separation technology. The AffiAmino UltraRapid Agarose magnetic agarose beads show outstanding magnetic behavior and are easily attracted to external magnets allowing separation within seconds. The agarose matrix enables minimal nonspecific binding of proteins due to its hydrophilic nature. The dark beads are easily observed by the naked eye, making them easy to follow and collect. The beads do not aggregate. The patented coupling technology allows rapid binding of ligands to the beads under mild conditions in water-based media. The beads bind 0.5-1 mg of human IgG per ml bead suspension (5-10 mg per ml settled beads), 0.5-0.75 mg Protein A per ml bead suspension (5-7.5 mg per ml settled beads) and similar amounts of other proteins. The bond is very stable (6 ppm leakage of Protein A) and the beads can be stored with bound ligand. The quantity of beads needed can easily be scaled up or down to match target protein concentration and sample volumes. The beads are suitable for separations from µl to 500 ml scale using appropriate magnetic separators, such as the LoabSep series. Product data Matrix Product Super-paramagnetic agarose AffiAmino UltraRapid Agarose, 10% bead suspension Particle size 45 µm 165 µm Coupling to Primary amino groups multipoint attachment Coupling conditions PBS (137 mm NaCl, 2.7 mm KCl, 10 mm phospate, ph 7.4; or similar recipe, e.g., 15 mm phosphate ph 7.4, 150 mm NaCl) Binding capacity* Storage conditions 0.5-1 mg human IgG / ml 10% bead suspension 5-10 mg human IgG / ml settled beads Store at 2-8 C in PBS with 20% ethanol Stability information** Stable for 6 months * Binding capacity was determined by incubating 1 ml 10% AffiAmino UltraRapid Agarose with human IgG (1 mg/ml in 1 ml PBS) for 60 minutes at room temperature. ** Data of product stability is continuously updated based on ongoing stability studies. 3

2. Principle of action AffiAmino UltraRapid Agarose beads are mixed with the protein intended for coupling. During a short incubation the protein is covalently coupled to the beads, which are then separated from solution using an external magnet. The coupled beads are then washed to remove excess protein and subsequently treated to block any remaining reactive groups. The coupled beads can be stored or used directly for affinity purifications, immunoprecipitations, etc. 3. Material supplied AffiAmino UltraRapid Agarose supplied as a 10% bead suspension in 15 mm phosphate ph 7.4, 150 mm NaCl, including 20% ethanol. 1 piece of Neodymium cube magnet (12 mm) suitable for separations in 0.5-5 ml volumes. 4. Additional materials needed Coupling/Washing buffer For coupling of proteins to beads and for washing, PBS (137 mm NaCl, 2.7 mm KCl, 10 mm phospate, ph 7.4) Blocking buffer To block remaining reactive groups on the beads use Ethanol amine (50 vol% in binding buffer) Binding buffer For binding of target protein to the functionalized beads use PBS PBS (137 mm NaCl, 2.7 mm KCl, 10 mm phospate, ph 7.4) Elution buffer For release of target protein from beads, 60 mm citric acid ph 2.2 to 3.0. Neutralization buffer To neutralize eluted proteins use, 1M Tris-HCl, ph 9 Storage buffer To store beads use PBS (137 mm NaCl, 2.7 mm KCl, 10 mm phospate, ph 7.4) with 20% ethanol Mixer or vortex to homogenize sample and beads. Gentle manual inversion of vial can also be used. Magnetic separator For separations in volumes larger than 5 ml use LoabSep 15/50 or LoabSep 500. Additional vials/tubes, pipets and pipet tips. 5. General handling Homogenize the bead suspension by using a vortex or by manual inversion, before dispensing the magnetic bead suspension into a test tube. Use a magnetic separator to attract the magnetic agarose beads to the wall of the test tube before each liquid removal step. Remove liquid carefully, trying not to disturb the magnetic beads. To avoid sample loss, make sure that no beads are accidently removed. In absence of the magnetic separator, add liquid and re-suspend the agarose beads by vortexing or by manual inversion. 4

6. Coupling and Binding Protocol 1. Equilibration for coupling Dispense the affinity magnetic beads in a test tube. 1 ml of 10% well suspended beads (100 µl bead volume (BV)) are suitable to couple 0.5-1mg protein. Remove the storage solution by magnetic separation. Re-suspend the magnetic beads in 10 bead volumes (BV) of binding buffer. Remove the liquid by magnetic separation. Re-suspend the magnetic beads in binding buffer. 2. Coupling Dissolve amino-containing ligand (Protein, peptide etc.) at a concentration of 1-2 mg/ml in binding buffer. It is very important that the ligand does not have any amino-containing impurities or contain ammonium sulfate, as these would also bind to the reactive structures. Add the amino-containing ligand to the magnetic bead suspension, total volume approximately 10 BV, and allow coupling for 1 hour at room temperature on vortex/mixing. Coupling efficiency of proteins and peptides could be determined by A280 measurements of the ligand solution before and after coupling to beads. 3. Washing Remove coupling solution from beads by magnetic separation. Wash out unbound ligand with 10 BV binding buffer. Repeat washing step twice. 4. Blocking Remaining reactive groups on beads are blocked with ethanol amine. Add typically 80 µl ethanol amine (50 vol% in binding buffer) to 1 ml bead suspension, 10% beads. Allow blocking for 45 minutes at room temperature on vortex/mixing. Wash with 10 BV binding buffer and repeat washing step once. Resuspend beads in 9 BV binding buffer, 10% medium, if beads are used within the following days. For longer storage resuspend beads in 20% ethanol solution. 5. Equilibration for binding Remove the storage solution by magnetic separation. Re-suspend the magnetic beads in 10 BV of binding buffer. Remove the liquid by magnetic separation. 5

6. Binding of target protein Note: Conditions for this and following steps depend on the bound ligand. Literature references might be consulted. Add target protein sample (diluted in e.g. binding buffer) and allow coupling for 1 hour at room temperature on vortex/mixing. Remove and collect the non-bound fraction by magnetic separation. 7. Washing Remove sample solution from beads by magnetic separation. Wash out unbound sample with 10 BV binding buffer. Repeat washing step twice. 8. Elution of target protein Add 3-10 BV of elution buffer and mix the solution at room temperature by vortexing or with gentle manual inversion of the test tube. After 15 minutes remove and collect the elution fraction, which contains the main part of the target protein Repeat the elution step if required, e.g. if low amount of target protein is obtained in the first elution step. 7. Practical notes Instead of using a magnetic separator in order to separate beads from solution in the washing steps, beads can also be washed on a sintered filter funnel Por 2 or 3. 1 ml of 10% well suspended (100 µl bead volume) beads are suitable to couple 0.5-1 mg protein or peptide. Beads caught in the lid or on the walls of the reaction vial/test tube can be removed by pipetting. If the magnetic separation of the magnetic beads are insufficient or beads are lost when washing use less beads or a stronger magnetic separator. If low amount of target protein is recovered increase the amount of magnetic beads or increase the time of coupling. If beads are aggregated between uses, vortex the bead suspension vigorously before next use. 8. Disclaimer The product is not fully tested and is not intended for human use. For in-vitro and research use only. 6

9. Ordering information Products Quantity Product no. AffiAmino UltraRapid Agarose 2x1 ml 10% beads 1003-2ml AffiAmino UltraRapid Agarose 5x1 ml 10% beads 1003-5ml AffiAmino UltraRapid Agarose 10 ml 10% beads 1003-10ml AffiAmino UltraRapid Agarose 50 ml 10% beads 1003-50ml Related products Quantity Product no. Protein A UltraRapid Agarose 2x1 ml 10% beads 1001-2ml Protein A UltraRapid Agarose 5x1 ml 10% beads 1001-5ml Protein A UltraRapid Agarose 10 ml 10% beads 1001-10ml Protein A UltraRapid Agarose 50 ml 10% beads 1001-50ml NdFeB cube magnet 1 2001 LoabSep 15/50 1 3001 LoabSep 500 1 4001 7

LAB ON A BEAD AB Postal address: Toftebergsvagen 7 SE-442 75 Lycke, Sweden Phone: +46 (0)70 513 01 15 Visiting address: Virdings Allé 28 SE-754 50 Uppsala, Sweden Phone: +46 (0)70 441 93 30 Email: info@labonabead.se Web: www.labonabead.se 8