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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 amines can be used directly.
Magnetic Beads Preparation
1.Prepare magnetic beads with 100% acetone (30 mg/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 3-4 times with the coupling buffer.
4. Remove the supernatant, and the washed beads are ready for coupling.
Peptide Coupling
1. The ligand concentration should be at least 200 μmoles per ml for small peptides. Mix the peptide sample (200 μmoles/ml) and the washed magnetic beads thoroughly by gentle rotation for 4-6 hours or overnight 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 reaction stop buffer containing 1M Glycine and incubate for 30-60 minutes at room temperature with gentle rotation.
4. Wash the beads 3-4 times with 1ml Washing Solution (or 1 M NaCl, 0.05% NaN3) by magnetic separation.
5. Add 0.5-1ml blocking buffer (PBS can also block beads, pH7.4, 0.1% BSA) to the beads and incubate at room temperature for 1 hour or at 4 °C overnight.
6. Wash the beads with 1ml of cold Wash buffer 3 times. Resuspend the beads in PBS buffer, pH 7.4, 0.1% BSA, and 0.01% azide (w/v) to desired concentration and store at 4°C until use. Do not freeze.
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 5-bed volumes of PBS buffer by vortex for 30 seconds. Leave the tube at room temperature for 1-3 minutes. Remove the supernatant using the magnetic separator (not provided).Repeat step 2 two times.
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.
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