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Sample Preparation
1. Dissolve 1-10mg protein/peptide in 1 ml coupling buffer. Protein concentration is typically 1-10mg/ml.
2. If samples have already been suspended in other buffers, dilute samples with an equal volume of coupling buffer.
3. Synthetic peptides with free sulfhydryls can be used directly. For large proteins, treat protein with 5-10 mM TCEP solution for 30 minutes at room temperature, followed by dialysis or a desalting column. For IgG antibody, use 2-Mercaptoethylamine•HCl (2-MEA).
Magnetic Beads Preparation
1.Suspend the magnetic beads with 20% Ethanol (Concentration: 30mg/ml).
2.Transfer and wash the beads by adding 1 ml coupling buffer and vortexing for 1-2 minutes.
3. Use a magnetic separator to separate and wash the beads 1-2 times with the coupling buffer.
Peptide Coupling
1. Mix the peptide sample and the magnetic beads thoroughly by gentle rotation for 60 minutes at room temperature.
2. Wash the magnetic beads with 1ml Coupling buffer four times using the magnetic separator (not provided).
3. Block the excess active groups on the beads by suspending the beads in 1ml Coupling buffer containing 8mg L-Cysteine•HCl and incubate for 30-60 minutes at room temperature with gentle rotation.
4. Wash the beads 3-4 times with 1ml Washing Solution (1 M NaCl, 0.05% NaN3) by magnetic separation.
5. Suspend the beads with the desired volume of coupling buffer, PBS, or any buffer compatible with the attached protein. Store at 4°C until ready for use.
Affinity Purification Protocol
1. It is recommended to titrate the number of beads for each application based on the amount of the target protein/antibody/peptide in your crude sample. Each mg of conjugated
magnetic beads normally binds to 1-20 µg target protein/peptide/antibody.
2. Wash the magnetic beads with 1ml Coupling buffer four times using the magnetic separator (not provided).
3. Add washed beads to the crude sample containing target protein/peptide/antibody and incubate at room temperature or desired temperature for 1-2 hours (room temperature) or overnight (4 C).
4. Extensively wash the beads with 5-bed bead volumes of PBS buffer or 1M NaCl until the absorbance of elute at 280 nm approaches the background level (OD 280 < 0.05).
5. 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 SDS-PAGE loading buffer.
Release the thiol-containing ligand from magnetic beads
1. Resuspend the magnetic beads with 0.1 M DTT (dithiothreitol) or Mercaptoethanol solution and incubate at room temperature for 30 minutes with gentle rotation.
2. Place the tube on the magnetic separator for 1-3 minutes. 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 the desired buffer.
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