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Sample Preparation
1. Dissolve 1-10 mg of protein or peptide in 1 ml of coupling buffer. The typical protein concentration is 1-10 mg/ml.
2. If your samples are suspended in other buffers, dilute them with an equal volume of coupling buffer.
3. Synthetic peptides containing free sulfhydryl groups can be used directly. For large proteins, treat them with a 5-10 mM TCEP solution for 30 minutes at room temperature, then follow with dialysis or use a desalting column. For IgG antibodies, use 2-mercaptoethylamine•HCl.
Magnetic Beads Preparation
1.Suspend the magnetic beads in ethanol (concentration: 30 mg/ml).
2.Transfer and wash the beads by adding 1 ml of coupling buffer and vortexing for 1-2 minutes.
3. Use a magnetic separator to separate the beads and wash them 1-2 times with the coupling buffer.
Peptide Coupling
1. Mix the peptide sample with the washed magnetic beads thoroughly by gentle rotation for 4-6 hours at room temperature with continuous rotation.
2. Wash the magnetic beads with 1 ml of coupling buffer four times using the magnetic separator.
3. Block the excess active groups on the beads by suspending them in 1 ml of coupling buffer containing 8 mg of L-Cysteine•HCl and incubate for 30-60 minutes at room temperature with gentle rotation.
4. Wash the beads 3-4 times with 1 ml of washing solution (1 M NaCl, 0.05% NaN3) using magnetic separation.
5. Suspend the beads in the desired volume of coupling buffer, PBS, or any compatible buffer containing 0.05% NaN3 with the attached peptide/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 target protein, antibody, or peptide in your crude sample. Typically, each mg of conjugated magnetic beads binds to 1-20 µg of target protein/peptide/antibody.
2. Wash the magnetic beads with 1 ml of coupling buffer four times using the magnetic separator.
3. Add the washed beads to the crude sample containing the target protein, peptide, or antibody, and incubate at room temperature for 1-2 hours or overnight at 4°C.
4. Extensively wash the beads with 5 bed volumes of PBS buffer or 1M NaCl until the absorbance of the elute at 280 nm approaches the background level (OD 280 < 0.05).
5. Elute the target protein using 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.
Cleaving the Disulfide Bond
Incubate the magnetic beads (30 mg/ml) in either 140 mM β-mercaptoethanol or 5 mM DTT.
a. Use 100 mM Tris-HCl (pH 8.0), 50 mM EDTA, and 140 mM β-mercaptoethanol for 2 hours to overnight at room temperature, or at 98°C for 5 minutes.
b. Use 100 mM Tris-HCl (pH 8.0), 50 mM EDTA, and 5 mM DTT for 2 hours to overnight at room temperature, or at 98°C for 5 minutes.
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