1. Dissolve 1-10mg protein/peptide in 0.5-1ml coupling buffer. Protein concentration is typically 1-10mg/ml.
2. If samples have already suspended in other buffers, dilute samples with an equal volume of coupling buffer.
1. Gently mix the magnetic beads thoroughly before use by shaking.
2. Place 1 mg of magnetic beads into a microcentrifuge tube.
3. Place the tube into a magnetic stand, collect the beads, and discard the supernatant.
4. Wash the beads three times with Washing Solution (1 ml each time) by magnetic separation, and re-suspend the beads in 0.5 ml of Coupling Solution.
5. Add 0.5 ml of the protein solution to 0.5 ml of the bead suspension; mix thoroughly and incubate for 4-16 hrs at 4°C on a rotator.
6. Add 2 ml of Blocking Solution. Mix thoroughly and incubate overnight at 4 °C on a rotator.
7. Collect the beads with a magnet and save the supernatant for analysis if needed.
8. Wash the protein-coupled beads twice with Washing Solution (1 ml each time) by magnetic separation.
9. Suspend the beads with the desired volume of Washing Solution or in a buffer compatible with the attached protein. Store at 4°C until ready for use.
1. Maleimides react with free sulfhydryl groups at a pH 6.5-7.5, forming stable, covalent thioether bonds. At pH > 7.5, maleimide reacts with amine groups. Therefore, accurate pH measurement is necessary when preparing solutions used in the experiment.
2. The reduction of disulfide bonds in the protein is needed before coupling. A 10-fold molar excess of a reducing agent such as DTT or TCEP is sufficient. If DTT is used, dialysis is required to remove excess DTT before the coupling step. However, the removal of TCEP is not needed.
3. EDTA can be included in the coupling buffer to chelate stray divalent metals that otherwise promote oxidation of non- reactive sulfhydryls.
4. Protein and bead concentrations should be optimized. Too low a protein concentration may result in bead crosslinking.
5. Do not freeze, dry, or centrifuge at high speed during the use or storage of beads. Otherwise, this may decrease the
binding capacity of the beads.