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Cat. No.
Product Name
Unit Size
Order
FE109
BcMag™ Aldehyde-Activated Magnetic Beads Conjugation Buffer Kit
Kit Components
– Conjugation Buffer: 100 ml (0.1 M sodium phosphate, pH 7.0)
– Blocking Buffer: 25 ml (1 M Tris•HCl, pH 7.4)
– 10x Wash Buffer: 25 ml (1 M NaCl)
– Coupling Reagent: 0.5 ml [5M NaCNBH3 (Dissolve in 1 M NaOH)]
Each
Specification
Composition
Magnetic beads are grafted with a high density of aldehyde groups 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
~260 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
~160 μmole / g of Beads
Storage
Ship at room temperature. Store at -20°C upon receipt.
The Aldehyde-Activated resins showcase superior coupling efficiency in comparison to cyanogen bromide (CNBr) activated supports. In addition, the aldehyde Chemistry results in an uncharged connection with the amine-containing ligand, which is more stable than the CNBr method. The hydrophilic surface of the Aldehyde-Activated resins ensures excellent dispersion and straightforward handling in various buffers. By utilizing these resins for affinity purification techniques, it results in better leak-resistant immobilization and reduced nonspecific binding. BcMag™ Aldehyde-Activated Magnetic Beads are appropriate for conjugating large proteins, while the BcMag™ Long-arm Aldehyde-Activated Magnetic Beads are the preferred choice for conjugating small peptides because the long-arm hydrophilic linker can decrease steric hindrance.
Protocol
Note:
●
This protocol can be scaled up as needed. We strongly recommended titration to optimize the number of beads used for each application.
Materials Required
1.
Coupling Buffer
Note:
●
The ionic strengths of the coupling buffers are critical to obtaining a high coupling efficiency rate.
The coupling buffers should be at minimal ionic strengths and should not contain any amino (e.g. Tris) or other nucleophiles. But the wash or storage buffers can contain amino.
●
Prepare buffer solution in a chemical fume hood because sodium cyanoborohydride is very toxic.
a.
Soluble ligand coupling buffer : 0.1 M sodium phosphate, pH 7.0
b.
b. Insoluble ligand coupling buffer: 0.1 M sodium phosphate, pH 7, 10-30% acetone, or dioxane, or alcohols, or dimethylformamide (DMF), or dimethylsulfoxide (DMSO) or 6 M Guanidine•HCl or 4 M Urea
2.
Blocking Buffer: 1 M Tris•HCl, pH 7.4
3.
Washing Buffer: 1 M NaCl
4.
Sodium Cyanoborohydride Solution (5 M): NaCNBH3 (MW 62.84) dissolved in 1 M NaOH
5.
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).
A.
Magnetic Beads Preparation
1.
Prepare 3% magnetic beads with acetone (30 mg/ml) and mix well.
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 using the coupling buffer, use the Bead as soon as possible due to the stability of the functional group.
B.
Protein coupling
1.
Dissolve 0.5-10mg protein/peptide in 1ml soluble coupling buffer if soluble. If insoluble, dissolve in 1ml Insoluble coupling buffer. If samples have already been suspended in other buffers, dilute samples with a 4-fold volume of coupling buffer or desalt or dialyze to buffer-exchange into coupling buffer.
Note:
●
Coupling efficiencies to Aldehyde-activated magnetic beads vary from ligand to ligand. The user should empirically optimize the concentration of the ligand. We recommend 0.5-10 mg/ml for protein conjugation and at least 200 μmoles ligands per ml for small peptides.
2.
Add 100μl of protein solution and 1μl NaCNBH3 solution (10μl per milliliter of total volume, final concentration 50 mM) to the washed beads in a fume hood. Resuspend the magnetic beads and mix well by pipetting and incubate the reaction at room temperature or 4°C overnight with continuous rotation.
3.
Wash beads three times with 1 ml coupling buffer.
4.
Add 0.5 blocking buffer and 5μl NaCNBH3 solution (10μl per milliliter of total volume, final concentration 50 mM) to the beads and incubate in the room for 1 hour or 4 °C overnight.
5.
Wash beads 4-6 times with 1 ml washing buffer.
C.
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.
●
Strongly recommend performing a 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 will cause higher backgrounds, while too few will cause lower yields. Each mg of conjugated magnetic beads 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 pipetting. 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 temperature or desired temperature for 1-2 hours (Lower temperature require longer incubation time).
Note: We strongly recommend performing 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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