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Sample Preparation
1. Dissolve 1-10 mg of protein/peptide in 1 ml of coupling buffer. A protein concentration of 1-10 mg/ml is typical.
2. If samples are suspended in other buffers, dilute them with an equal volume of coupling buffer.
3. Synthetic peptides with free sulfhydryls can be used directly. For large proteins, treat them with a 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.Prepare magnetic beads with 100% acetone (30 mg/ml). Note: Store unused beads in acetone solution at 4°C.
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 and wash the beads 1-2 times with the coupling buffer.
Peptide Coupling
1. Mix the peptide sample and magnetic beads thoroughly by gentle rotation in the dark (wrap with aluminum foil) for overnight incubation at room temperature.
2. Wash the magnetic beads with 1 ml of coupling buffer four times using the magnetic separator (not provided).
3. Block excess active groups on the beads by suspending the beads 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 to 4 times with 1 ml of Washing Solution (1 M NaCl) using magnetic separation.
5. Suspend the beads in the desired volume of coupling buffer, PBS, or any buffer containing 0.05% sodium azide that is compatible with the attached protein. Store at 4°C until ready for use.
Affinity Purification Protocol
1. It is advisable to titrate the number of beads for each application based on the amount of the target protein, antibody, or peptide present in your crude sample. Typically, each mg of conjugated magnetic beads can bind to 10-20 µg of target protein, peptide, or antibody.
2. Wash the magnetic beads with 1 ml of coupling buffer four times using a magnetic separator (not provided).
3. Add the washed beads to the crude sample containing the target protein, peptide, or antibody. Incubate at room temperature for 1-2 hours or overnight at 4°C.
4. Wash the beads extensively with 5 bed volumes of PBS buffer or 1 M NaCl until the absorbance of the eluent 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.
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