Amine-activated Peptide Conjugation Magnetic Beads

The magnet is not provided.

Coupling capacity was determined by incubating 30mg beads with human lgG (1 mg/ml in 1ml PBS) for 60 minutes at room temperature.

Solvent: 20% ethanol

5% Glutaraldehyde solution: add 5ml of 25% aqueous glutaraldehyde solution to 20ml of Coupling Buffer.

Coupling Solution: 10mM pyridine (Add 0.8ml of pyridine to 900ml of water. Adjust the pH to 6 with HCL. Add water to a final volume of 1L.)

Quenching Solution: 1.0M glycine (Dissolve 7.5g of glycine in 90ml of water. Adjust the pH to 8 with 10N NaOH. Add water to a final volume of 100ml.)

Washing Buffer: 0.01 M Tris base containing 0.15 M NaCl, 0.1% (w/v) BSA, 0.001M EDTA, sodium salt and 0.1% (w/v) sodium azide (Dissolve 1.21g of Tris base, 8.7g of NaCl, 1g of BSA, 0.37g of EDTA, sodium salt and 1.0g of sodium azide in 900ml of water. Adjust the pH to 7.4 with HCl. Add water to a final volume of 1L.)

Sample Preparation

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.

Bead activation

1. Gently mix the magnetic beads thoroughly before use by shaking.

2. Place 1mg of magnetic beads into a microcentrifuge tube.

3. lace the tube into a magnetic stand, collet the beads, and discard the supernatant.

4. Wash the beads three times with Coupling Buffer (1ml each time) by magnetic separation.

5. Re-suspend the beads by adding 400ul of 5% Glutaraldejhude and shake. Incubate at room temperature for 3 hrs with gentle rotation.

6. Separate beads using a mganet, remove the supernatant.

7. Wash beads three times with 1ml Coupling Buffer to remove unreacted glutaraldehyd.

Protein Coupling

1. Add 200ul protein solution into the tube containing activated beads and mi well by shaking. Incubate for 24 hrs at room temperature or 4°C with gentle rotation.

2. Separate beads using a magnet, remove the supernatant.

3. Add 800ul of Quenching Solution into the tube. Shake to suspend the beads. Gently shake for 30min at room temperature.

4. Wash the beads with a 1ml Washing buffer three times.

5. Suspend the beads with the desired volume of washing buffer or in a buffer compatible with the attached protein. Store at 4°C until ready for use.

Important Notes

1. This protocol can be used for both coupling primary amine-containing and carboxy-containing ligands.

2. The coupling buffer should not contain any amino (e.g., Tris) or carboxyl groups (e.g., acetate and citrate).

3. The solutions containing glutaraldehyde or pyridine are volatile and harmful. Please perform operations with these solutions in a chemical fume hood.

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.