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Cat. No.
Product Name
Unit Size
Order
FI109
BcMag™ NHS-Activated Magnetic Beads Conjugation Buffer Kit
Kit Components
– 10x Coupling Buffer: 15 ml (100 mM potassium phosphate, 1.5 M NaCl, pH 5.5)
– 1x Block Buffer: 10 ml (1.0 M Ethanolamine, pH 9)
– 10x Wash Buffer: 5 ml (0.5 M Tris-HCl, 5 M NaCl, pH 8.0)
Each
Specification
Composition
Magnetic beads grafted with NHS 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 3-11; Long-Term: pH 4-10
Temperature: 4°C -140°C; Most organic solvents
Magnetization
~40-45 EMU/g
Type of Magnetization
Superparamagnetic
Formulation
Lyophilized Powder
Functional Group Density
1μm Magnetic Beads
~250 μmole / g of Beads
5μm Magnetic Beads
~200 μmole / g of Beads
1μm Long-Arm Magnetic Beads
~210 μmole / g of Beads
5μm Long-Arm Magnetic Beads
~170 μmole / g of Beads
Storage
Store at -20°C, free of moisture upon receipt
The amine group (-NH2) is commonly used to attach protein molecules to surfaces. This group can be found at the beginning of each protein chain and in certain amino acids. Because these groups are positively charged in normal conditions, they are usually found on the outside of proteins and are easy to attach without damaging the protein structure.
BcMag™ NHS-Activated Magnetic Beads are magnetic beads coated with NHS functional groups on the surface. These beads use safe chemistry to attach ligands with primary amine groups to the surface, forming stable linkages. This process is efficient and can be completed quickly. BcMag™ Long-arm NHS-activated Magnetic Beads are recommended for smaller peptides to reduce steric hindrance. These magnetic beads can be used in various affinity purification methods.
Protocol
Note:
●
This protocol can be scaled up as needed. We strongly recommended titration to optimize the number of beads used for each application.
●
Avoid tris or other buffers containing primary amines because these will compete with the intended coupling reaction.
A.
Materials Required
1.
Magnetic Rack (for manual operation)
Based on sample volume, the user can choose one of the following Magnetic Racks:
1. BcMag™ Magnetic Rack-2 for holding two individual 1.5 ml centrifuge tubes (Cat. No. MS-01);
2. BcMag™ Magnetic Rack-6 for holding six individual 1.5 ml centrifuge tubes (Cat. No. MS-02);
3. BcMag™ Magnetic Rack-24 for holding twenty-four individual 1.5-2.0 ml centrifuge tubes (Cat. No. MS-03);
4. 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);
5. 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, 0.15 M NaCl, pH 7.4
3.
Wash Buffer: 0.05 M Tris-HCl, 0.5 M NaCl, pH 8.0
4.
Blocking Buffer: 1 M ethanolamine, pH 9
B.
Beads preparation
1.
Prepare 3% magnetic beads with 100% acetone (30 mg/ml).
Note: Store the unused beads in acetone 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.
C.
Coupling
Note:
●
Coupling efficiencies to NHS-activated magnetic beads vary from ligand to ligand. The user should empirically optimize the concentration of the ligand. The ligand concentration should be at least 200 μmoles per ml for small peptides. 0.5-10 mg/ml is recommended for protein conjugation.
1.
Prepare 100 μl of protein solution (0.5-1mg/ml) or peptide solution (200 μmoles/ml) with coupling buffer and mix with the above-washed beads. If samples have already been suspended in another buffer, dilute samples with an equal volume of coupling buffer.
2.
Incubate the reaction with continuous rotation at room temperature for 4-6 hours or overnight.
Note: The user should optimize the incubation time.
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 wash buffer by vortex for 30 seconds. Place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack.
4.
Wash beads 3-4 times with 1 ml wash buffer (or 1 M NaCl).
5.
Add 0.5-1ml blocking buffer (PBS can also block beads, pH7.4, 0.1% BSA) to the beads and incubate at room temperature for 1 hour or at 4 °C overnight.
6.
Wash the beads with 1ml of cold Wash buffer 3 times.
Resuspend the beads in PBS buffer, pH 7.4, 0.1% BSA, and 0.01% azide (w/v) to desired concentration and store at 4°C until use. Do not freeze.
D.
General Affinity Purification Protocol
Note:
●
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.
●
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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