- +1 858 909 0079
- +1 858 909 0057
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- +1 858 909 0079
- [email protected]
Cat. No.
Product Name
Unit Size
Order
Specification
Composition
Magnetic beads grafted with tosyl group on the surface
Number of Beads
~ 1.68 x 109 beads/mg (1μm beads)
~ 5 x 107 beads /mg (5μm beads)
Stability
Short Term (<1 hour): pH 4-11; Long-Term: pH 4-10
Temperature: 4°C -140°C; Most organic solvents
Magnetization
~40-45 EMU/g
Formulation
Lyophilized Powder
Functional Group Density
1μm Magnetic Beads
~250 μmole / g of Beads
5μm Magnetic Beads
~210 μmole / g of Beads
1μm Long-Arm Magnetic Beads
~200 μmole / g of Beads
5μm Long-Arm Magnetic Beads
~180 μmole / g of Beads
Storage
Ship at room temperature. Store at -20°C, free of moisture upon receipt.
Tosyl Activated Magnetic Beads are special beads that have tosyl functional groups on their surface. These beads are excellent at binding ligands that have primary amine, and they can do this efficiently in both aqueous and organic solvents (30% DMF) without any charge. Short-arm and long-arm tosyl-activated magnetic beads have different properties, with short-arm beads being better suited for conjugating large-sized proteins while long-arm beads are ideal for small molecules that don’t have steric hindrance problems. You can use Tosyl Activated magnetic resins to covalently attach antibodies, peptides, complete proteins, and functional enzymes to the bead surface. Many researchers use these immobilized beads for Immunoprecipitation of proteins and protein complexes because they don’t interfere with the experiment, and they bind antibodies covalently to the bead surface.
When using Tosyl Activated magnetic resins, the coupling reaction should be carried out at 37°C, and the pH should be neutral to high. It’s best to aim for a pH of 8.5-9.5 for coupling, but if the ligand is sensitive to pH, you can use an alternate buffer at pH 7.4.
The unique dry form eliminates the need for acetone solvent storage or removal and disposal. Furthermore, because the dry resin concentrates the sample as it swells, lowering the volume of the starting material and resulting in highly effective ligand immobilization, it is perfect for coupling reactions with dilute materials.
The tosyl-activated magnetic beads work perfectly as solid support for various bio separations to refine molecules, cells, and parts of cells into purified fractions. After conjugation with ligands, add the beads to a solution containing the target molecules, then mix, incubate, wash and elute the target molecules
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Pre-activated and ready-to-use
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Easy to use
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No charge remains on the surface after coupling
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Stable covalent bond with minimal ligand leakage
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Produces reusable immunoaffinity matrices
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Low nonspecific binding
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Immobilize 1-10 mg protein or 0.1-1 mg peptide/ml beads
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Applications: Cell sorting, Immunoprecipitation, purification for antibodies, proteins/peptides, DNA/RNA
Protocol
Note:
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This protocol can be scaled up as needed. We strongly recommended titration to optimize the number of beads used for each application.
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Avoid tris or other buffers containing primary amines because these will compete with the intended coupling reaction.
Materials required
1.
Magnetic Rack (for manual operation)
Based on sample volume, the user can choose one of the following Magnetic Racks:
– BcMag™ Magnetic Rack-2 for holding two individual 1.5 ml centrifuge tubes (Cat. No. MS-01);
– BcMag™ Magnetic Rack-6 for holding six individual 1.5 ml centrifuge tubes (Cat. No. MS-02);
– BcMag™ Magnetic Rack-24 for holding twenty-four individual 1.5-2.0 ml centrifuge tubes (Cat. No. MS-03);
– BcMag™ Magnetic Rack-50 for holding one 50 ml centrifuge tube, one 15 ml centrifuge tube, and four individual 1.5 ml centrifuge tubes (Cat. No. MS-04);
– BcMag™ Magnetic Rack-96 for holding a 96 ELISA plate or PCR plate (Cat. No. MS-05).
2.
Coupling Buffer: 0.1 M sodium phosphate, pH 7.4
Note:
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The coupling buffers should be minimal ionic strengths and should not contain any amino (e.g., Tris or glycine). But the wash or storage buffers can have amino or carboxyl groups
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Water-insoluble ligands can be conjugated in 30% organic solvent (30% DMF) with a coupling buffer.
3.
Blocking Buffer: PBS pH 7.4 with 0.5% (w/v) BSA
4.
Washing buffer: PBS pH 7.4 with 0.1% (w/v) BSA.
A.
Magnetic Beads Preparation
1.
Prepare 3% magnetic beads with 100% isopropanol (30 mg/ml).
Note: Store the unused beads in isopropanol solution at 4°C. It is stable for over a year.
2.
Transfer 100 μl (3mg) magnetic beads to a centrifuge tube.
3.
Place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack. Remove the tube from the rack and resuspend the beads with 1 ml coupling buffer by vortex for 30 seconds.
4.
Repeat step 3 two times.
5.
Remove the supernatant, and the washed beads are ready for coupling.
Note: Once rehydrated, use the bead as soon as possible due to the stability of the functional group.
B.
Protein Coupling
1.
Prepare 100 μl of protein solution (0.5-1mg/ml) or peptide solution (200 μmoles/ml) with coupling buffer.
Note: Coupling efficiencies vary from ligand to ligand. The user should empirically optimize the concentration of the ligand.
2.
Add the protein or peptide solution to the washed beads and mix well by vortex or pipette.
3.
Incubate the reaction for 24-48 hours at 20-25°C or 48-72 hours at 4°C with continuous rotation.
4.
Wash beads three times with 1 ml washing buffer.
5.
Add 1ml blocking buffer to the beads and incubate at room for 1 hour or at 4 °C overnight.
6.
Wash beads 4-6 times with 1 ml PBS buffer.
7.
Resuspend the beads in PBS buffer with 0.01% azide (w/v) to desired concentration and store at 4°C until use. Do not freeze.
C.
General Affinity Purification Protocol
Note:
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This protocol is a general affinity purification procedure. Designing a universal protocol for all protein purification is impossible because no two proteins are precisely alike. To obtain the best results, each user must determine the optimal working conditions for the purification of the individual target protein.
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We strongly recommended titration to optimize the number of beads used for each application based on the amount of the target protein in the crude sample. Too many magnetic beads used will cause higher backgrounds, while too few beads used will cause lower yields. Each mg of magnetic beads typically binds to 10-20 μg of the target protein.
1.
Transfer the optimal amount of the beads to a centrifuge tube. Place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack.
2.
Remove the tube and wash the beads with 5-bed volumes of PBS buffer by vortex for 30 seconds. Leave the tube at room temperature for 1-3 minutes. Place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack.
3.
Repeat step 2 two times.
4.
Add washed beads to the crude sample containing the target protein and incubate at room or desired temperature for 1-2 hours (Lower temperatures require longer incubation time).
Note: Strongly recommended to perform a titration to optimize incubation time. More prolonged incubation may cause higher background.
5.
Note: Adding a higher concentration of salts, nonionic detergent, and reducing reagent may reduce the nonspecific background. For example, adding NaCl (up to 1-1.5 M), 0.1-0.5% nonionic detergents such as Triton X 100 or Tween 20, and a reducing reagent such as DTT or TCEP (we usually use 3mM) to the washing buffer.
6.
Elute the target protein by appropriate methods such as low pH (2-4), high pH (10-12), high salt, high temperature, affinity elution, or boiling in an SDS-PAGE sample buffer.
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