Magnetic Beads Preparation
1.Suspend the magnetic beads with 20% Ethanol (Concentration: 20mg/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 2-3 times with the coupling buffer.
4. Resuspend the magnetic beads by adding 20 ml of 5% Glutaraldehyde and shake vigorously. Leave at room temperature for 3 hr with gentle rotation.
5. Wash beads three times with 30ml coupling buffer as described to remove unreacted glutaraldehyde.
Coupling of Protein
For some expensive proteins, such as monoclonal antibodies, the supplied concentration cannot reach the required 2.5-10mg/ml. To ensure highly efficient coupling, the BSA should be added to the protein solution to bring protein concentration to the required level.
1. Prepare protein solution by adding 5-20 mg protein into 10 ml coupling buffer and mix very well.
2. Add the protein solution into the tube containing activated beads and Mix well by vigorously shaking.
Leave reaction for 24 hr at room temperature with gentle rotation.
3. When the reaction is finished, place the tube into the magnetic separator. Place the tube on the magnetic separator for 1-3 minutes. Remove the supernatant while the tube remains on the separator.
4. Add 40ml of reaction stop buffer into the tube. Shake vigorously to suspend the beads. Gently shake for 30 min at room temperature.
5. Wash the beads with 30 ml storage buffer three times.
6. Suspend the beads with the desired volume of storage buffer and store at 4ºC.
General Affinity Purification Protocol
1. Transfer the optimal amount of the beads to a centrifuge tube. Place the tube on the magnetic separator for 1-3 minutes. Remove the supernatant while the tube remains on the separator.
It is strongly recommended that a titration be performed to optimize the quantity of beads used for each application based on the amount of the target protein in a crude sample. Too many magnetic beads used will cause higher backgrounds, while too few beads used will cause lower yields. Each mg of conjugated magnetic beads normally binds to 1-20 µg target protein.
2. Remove the tube and resuspend the beads with a 5-bed bead volume of PBS buffer by vortex for 30 seconds. Leave the tube at room temperature for 1-3 minutes. Place the tube on the magnetic separator for 1-3 minutes. Remove the supernatant while the tube remains on the separator.
3. Repeat step 2 two times
4. Add washed beads to a crude sample containing target protein and incubate at room temperature or desired temperature for 1-2 hours (Lower temperature requires 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 the background level (OD 280 < 0.05).
6. 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.