Introduction
PuriMag? Thiol-activated Magnetic Beads are uniform, silica-based superparamagnetic beads coated with high density Thiol functional groups (2-pyridyl disulphide) on the surface. The beads are used to reversible couple thiol-containing ligands. PuriMag? thiol-activated Magnetic Beads are most suitable for conjugation of large proteins. The beads is used in various applications such as separate thiol-containing proteins and peptides from non-thiol containing proteins or peptides, purify enzyme with active thiol site from denatured enzymes and store and protect thiol containing proteins.
Features and Advantages:
· Pre-activated and ready-to-use
· Recommended coupling conditions: pH 4–8, 4°C to 25°C, 3–16 h.
· Specific isolation of cysteine proteins/peptides
· Stable covalent bond with minimal ligand leakage
· Produces reusable immunoaffinity matrices
· Low nonspecific binding
· Immobilize 5-50 mg protein or 0.5-5 mg peptide/g beads
· Applications: Cell sorting, Immunoprecipitation; Purification for Antibodies, Proteins/Peptides, DNA/RNA
Product specific
Group density: 25~35 μmoles 2-pyridyl disulphide /g beads
particle form：Superparamagnetic sphere
Bead diameter：0.2 μm (nominal)
Storage：10 mg/mL in 20% ethanol, 1 mM EDTA at 2~8 °C. Do not freeze.
The beads are shipped at ambient temperature. If stored and handled correctly the beads have one year shelf life.
Protocol
The following protocol is an example for coupling protein and/or peptides to PuriMag? Thiol -Activated magnetic beads. It is strongly recommended that a titration be performed to optimize the quantity of beads used for each individual application.
A. Materials Required
· Coupling Buffer : 0.1 M sodium phosphate, pH 7.0 , 5mM EDTA
· L-Cysteine·HCl
· TCEP (tris(2-carboxyethyl)phosphine)
· Washing Buffer: 1 M NaCl, 0.05% NaN3
· Magnetic Separator
Note: All buffers should be de-gased to avoid oxidation of free thiol groups. Tris, phosphate or acetate buffer systems with the addition of 0.1–0.5 M NaCl may be used, e.g. 0.1 M Tris-HCl, pH 7.5, 0.5 M NaCl. 1 mM EDTA may be added to remove trace amounts of heavy metal ions, which may catalyze oxidation of thiols.
B. Sample Preparation
Note: § Make sure that protein/peptide to be conjugated has free (reduced) sulfhydryls. To ensure free sulfhydryl groups available, disulfide bonds must be reduced with a reducing agent such as DTT (dithiothreitol), TCEP (tris(2- carboxyethyl)phosphine), or 2-MEA (2-Mercaptoethylamine·HCl) followed by desalting or dialysis to remove the reducing agent.
§ Newly Synthesized peptides may be directly used for coupling, if used immediately after reconstitution
§ For protein, treat protein with 5-10 mM TCEP solution for 30 mins at room temperature, followed by dialysis or desalting column. For IgG antibody, 2-MEA is recommended due to its Selective reduction of hinge-region disulfide bonds
§ If the sample contains reducing agents with free sulfhydryls (e.g., 2-mercaptoethanol, DTT or TCEP), these agents must be completely removed by dialysis or desalting.
1. Dissolve 1-10mg protein/peptide in 1ml coupling buffer.
2. If samples have already suspended in other buffer, dilute samples with equal volume of coupling buffer.
C. Magnetic Beads Preparation
Note: Weight, suspend the magnetic beads with 20% Ethanol (Concentration: 10mg/ml), disperse the beads by vigorously vortexing and store at 4?C. Shake the bottle to completely resuspend the Magnetic Beads before use.
1. Transfer 10 mg Magnetic beads to a centrifuge tube. Resuspend the beads by adding 1 ml coupling buffer and mix the beads by vigorous vortexing for 1-2 mins.
2. Place the tube on the magnetic separator for 1-3 mins. Remove the supernatant while the tube remains on the separator. Remove the tube from the separator and resuspend the beads with 1 ml coupling buffer by vortex for 30 s.
3. Repeat step-2 once.
D. Coupling
1. Add sample to the beads and incubate for 60 mins at room temperature with gentle rotation.
2. Washed the beads with 1ml Coupling buffer for four times.
3. Block the excess active groups on the beads by suspending the beads in 1ml Coupling buffer containing 8mg L-Cysteine·HCl and incubate 30-60 mins at room temperature with gentle rotation.
4. Wash the beads with 1ml Washing buffer four times as described in B3.
5. Resuspend the beads in PBS buffer containing 0.05% sodium azide and store at 4°C.
E. General Affinity Purification Protocol
1. Transfer optimal amount of the beads to a tube. Place the tube on the magnetic separator for 1-3 mins. Remove the supernatant while the tube remains on the separator.
Note: · It is strongly recommended that a titration be performed to optimize the quantity of beads used for each individual application based on the amount of the target protein in crude sample. Too many magnetic beads used will cause higher backgrounds, while too little beads used will cause lower yields. Each mg of conjugated magnetic beads normally bind to 1-20 μg target protein.
2. Remove the tube and resuspend the beads with 5 bed bead volume of PBS buffer by vortex for 30 s. Leave the tube at room temperature for 1-3 mins. Place the tube on the magnetic separator for 1-3 mins. Remove the supernatant while the tube remains on the separator.
3. Repeat step 2 two times
4. Add washed beads to crude sample containing target protein and incubate at room temperature or desired temperature for 1-2 h (Lower temperature require longer incubation time).
5. Extensively wash the beads with 5 bed bead volumes of PBS buffer or 1M NaCl until the absorbance of elute at 280 nm approaches background level (OD 280 < 0.05).
6. Elute the target protein by appropriated methods such as low pH (2-4), high pH (10-12), high salt, high temperature , affinity elution or boiling in SDS-PAGE loading buffer.
F. Release the thiol-containing ligand from magnetic beads
1. Resuspend the beads with 0.1 M DTT or Mercaptoethanol solution and incubate at room temperature for 30 mins with gentle rotation.
2. Place the tube on the magnetic separator for 1-3 mins. Remove the supernatant containing the released ligand to a new centrifuge tube while the tube remains on the separator.
3. Perform buffer change by gel filtration or dialysis to dissolve the ligand into desired buffer.
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