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Cat. No.
Product Name
Unit Size
Order
Specification
Composition
Magnetic beads coated with a high density of primary amine
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
Concentration
30 mg/ml (1 mM ETDA, pH 7.0)
Functional Group Density
1μm Magnetic Beads
~250 μmole / g of Beads
5μm Magnetic Beads
~200 μmole / g of Beads
1μm Long-Arm Magnetic Beads
~180 μmole / g of Beads
5μm Long-Arm Magnetic Beads
~135 μmole / g of Beads
Storage
Store at 4°C upon receipt. Do not freeze.
BcMag™ Amine-terminated Magnetic Beads are magnetic beads that possess a high concentration of primary amine functional groups on their surface. These functional groups enable the covalent conjugation of primary amine or carboxy-containing ligands, including proteins and peptides, for the purpose of affinity purification (as shown in Figure below). The hydrophilic nature of the bead surface leads to reduced levels of nonspecific adsorption and excellent dispersal properties, allowing for easy handling of the beads across various buffers. For larger proteins, BcMag™ Amine-terminated Magnetic Beads are deemed the most optimal option for conjugation. For small peptides, on the other hand, BcMag™Long-arm amine-terminated Magnetic Beads are suggested due to their long-arm (21-atom) hydrophilic linker, which reduces steric hindrance.
A process that utilizes carbodiimide mediation may be implemented to immobilize biological molecules with carboxylic acids. Although there are no activated supports that possess a reactive group capable of spontaneous reaction with carboxylates, chromatographic supports containing amines (or hydrazides) can be employed to create amide bonds with carboxylates that have been activated using the water-soluble carbodiimide crosslinker EDC.
In the workflow, BcMag™ Amine-Terminated magnetic beads serve as a solid resin that is well-suited for various affinity purifications. This is useful for refining molecules, cells, and parts of cells into purified fractions. Following conjugation with ligands, the beads are added to a solution containing the target molecules, then mixed, incubated, washed, and the target molecules are eluted (as illustrated in Figure below).
I.
Protocol (Carboxy-containing ligand conjugation)
Note:
●
The following protocol is an example for coupling carboxy-containing ligands to BcMag™ Amine-terminated magnetic beads. We strongly recommended performing a titration to optimize the number of beads used for each application. This protocol can be scaled up and down accordingly.
●
The ionic strengths of the coupling buffers are critical to obtaining a higher coupling efficiency rate.
●
The coupling buffers should have minimal ionic strengths and contain no amino (e.g., Tris) or carboxyl groups (e.g., acetate, citrate). But the wash or storage buffers can contain amino or carboxyl groups.
●
Conjugate water-insoluble ligands in a coupling buffer containing a final concentration of up to 50% purified dioxane or ethylene glycol. If a mixture of buffer solution and an organic solvent has been used, wash the final product with this mixture of buffer solution.
●
The washing procedure should be followed by a wash with distilled water and then a wash with the buffer in the affinity chromatography stage.
Materials Required
●
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).
●
Coupling Buffer: 10 mM potassium phosphate, 0.15 M NaCl, pH 5.5 or 0.1 M MES Buffer, 0.15 M NaCl, pH 4.5-5.5.
Note: For Long-arm carboxy-terminated magnetic beads, adjust NaCl concentration to 0.3-0.5 M (Final concentration) in coupling buffer.
●
EDC [1-ethyl-3 (3-dimethyaminopropyl) carbodiimide], Sigma, Cat. No. E7750
●
Wash/Storage Buffer: 10 mM Tris base, 0.15 M NaCl, 0.1% (w/v) BSA, 1mM EDTA, 0.01% sodium azide, pH 7.5.
●
Blocking buffer: 1 M Glycine, pH 8.0
Coupling
A.
Magnetic Beads Preparation
Note:
●
Shake or vortex the bottle to completely resuspend the magnetic beads before using.
●
Do not allow the magnetic beads to sit for more than two minutes before dispensing.
1.
Combine 30 mg with 1 ml coupling buffer and mix well by vortex or pipette.
2.
Insert the tube into a magnetic rack for 1-3 minutes until the supernatant becomes clear. Aspirate and discard the supernatant with a pipette while the tube remains in the rack.
3.
The beads are ready for coupling.
B.
Coupling
1.
Prepare protein solution (0.5-1mg/ml) with coupling buffer and mix with above-washed beads.
2.
Freshly prepare 2% EDC solution with coupling buffer. Note: use within 15 minutes of preparing.
3.
Add 100 μl of 2% EDC solution to the protein solution and mix well
4.
Incubate at room temperature or t at 4°C overnight with good mixing (end-over-end).
C.
Coupling
1.
When the reaction is finished, place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack.
2.
Washing the beads with 5 ml Wash/storage buffer three times.
3.
Incubate the beads with 1ml of Blocking buffer at room temperature with good mixing (end-over-end) for 1-2 hours
4.
Washing the beads with 5 ml Wash/storage buffer three times
5.
Suspend the beads with the desired Wash/storage buffer volume and store them at 4º C.
II.
Protocol (Amine-containing ligand conjugation)
Note:
●
The following protocol is an example of coupling primary amine-containing ligands to BcMag™ Amine-terminated magnetic beads. This protocol can be scaled up and down accordingly. It is strongly recommended that a titration be performed to optimize the quantity of beads used for each application.
●
The ionic strengths of the coupling buffers are critical to obtaining a higher coupling efficiency rate.
●
The coupling buffers should have minimal ionic strengths and contain no amino (e.g., Tris) or carboxyl groups (e.g., acetate, citrate). But the wash or storage buffers can contain amino or carboxyl groups.
A.
Buffer Preparation
Note:
Prepare buffer solution in a chemical fume hood because Glutaraldehyde or pyridine is volatile and toxic.
●
Coupling Buffer: 10 mM pyridine
●
5% Glutaraldehyde: Add 5.0 ml of 25% glutaraldehyde to 20 ml of Coupling Buffer.
●
Reaction Stop buffer: 1M Glycine.
●
Wash Buffer: 10 mM Tris base, 0.15 M NaCl, 0.1%(w/v) BSA, 1 mM EDTA, 0.01% sodium azide
B.
Bead activation
Note:
Shake the bottle to resuspend the Magnetic Beads entirely before use.
1.
Combine 30 mg with 1 ml coupling buffer and mix well by vortex or pipette.
2.
Insert the tube into a magnetic rack for 1-3 minutes until the supernatant becomes clear. Aspirate and discard the supernatant with a pipette while the tube remains in the rack.
3.
Resuspend the magnetic beads with 1 ml of 5% Glutaraldehyde and shake vigorously. Incubate at room temperature for 3 hours with good mixing (end-over-end).
4.
Place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack.
5.
Wash beads three times with 1ml coupling buffer to remove unreacted Glutaraldehyde.
C.
Coupling
Note:
For some expensive proteins, such as monoclonal antibodies, the supplied concentration cannot reach the
required 0.5-1 mg/ml. The BSA should be added to the protein solution to ensure highly efficient coupling to bring
protein concentration to the required level.
1.
Prepare 1 ml of protein solution (0.5-1mg/ml) with coupling buffer and mix with above-terminated beads very well by vortex or pipette.
2.
Incubate at room temperature or t at 4°C overnight with good mixing (end-over-end).
3.
When the reaction is finished, place the tube into the magnetic rack. Place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack.
4.
Add 1ml of reaction stop buffer into the tube. Shake vigorously to suspend the beads. Incubate at room temperature for 30 minutes.
5.
Washing the beads with 1 ml storage buffer three times.
6.
Suspend the beads with the desired volume of storage buffer and store at 4º C.
III.
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.
Extensively wash the beads with 5-beads volumes of PBS buffer or 1M NaCl until the absorbance of eluting at 280 nm approaches the background level (OD 280 < 0.05).
Note: Adding a higher concentration of salts, nonionic detergent, and reducing agents 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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