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.

Coupling Buffer: 10 mM pyridine
Add 800 µl pyridine to 900 ml of ddH2O. Adjust to pH 6.0 with HCl. Add ddH2O to 1 Liter

5% Glutaraldehyde:
Add 5.0 ml of 25% glutaraldehyde to 20 ml of Coupling Buffer.

Reaction Stop buffer: 1M Glycine
Dissolve 7.5 g Glycine in 90 ml of ddH2O. Adjust to pH 8.0 with 10N NaOH. Adjust the finalvolume to 100 ml with ddH2O

Wash Buffer: 10 mM Tris base, 0.15 M NaCl, 0.1%(w/v) BSA, 1 mM EDTA, 0.1% sodium azide
Dissolve 1.21g Tris base, 8.7g NaCl, 1.0 g BSA, 0.37g EDTA, sodium salt, 1.0 g sodium azide in 900ml ddH2O. Adjust to pH 7.4 with HCl. Adjust the final volume to 1 Liter with ddH2O.

Bead activation:

Weigh and suspend the magnetic beads with 1mM EDTA , pH 7.0 (Concentration: 30mg/ml) and store at 4C.

1. Transfer 1 ml of the beads to a 15ml tube. Place the tube on the magnetic separator for 1-3 minutes. Remove the supernatant while the tube remains on the separator.

2. Remove the tube and resuspend the beads with 10ml Coupling 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. Resuspend the magnetic beads by adding 15 ml of 5% Glutaraldehyde and shake vigorously. Leave at room temperature for 3 hr with gentle rotation.

5. Place the tube on the magnetic separator for 1-3 minutes. Remove the supernatant while the tube remains on the separator.

6. Wash beads three times with 15ml coupling buffer as described to remove unreacted glutaraldehyde.

Protein Coupling

1. Prepare protein solution by adding 1-10 mg protein into 10 ml coupling buffer and mix very well.

2. Add the protein solution into the tube containing activated beads (step 6 in Bead Preparation) 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 10ml of reaction stop buffer into the tube. Shake vigorously to suspend the beads. Gently shake for 30 min at room temperature.

5. Washing the beads with 10 ml storage buffer three times.

6. Suspend the beads with desired volume of storage buffer and store at 4º C.

General Affinity Purification Protocol
Note:
This protocol is a general affinity purification procedure. It is impossible to design a universal protocol for all protein purification because no two proteins are exactly alike. In order to obtain the best results, each user must determine the optimal working conditions for purification of the individual target protein.

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.
   Note: It is strongly recommended that titration is performed to optimize the number of beads used for each individual application based on the amount of the target protein in the crude sample. Too many magnetic beads used will cause higher backgrounds, while too little beads used will cause lower yields. Each mg of conjugated magnetic beads normally bind to 1-20 ug target protein.
2. Remove the tube and resuspend the beads with 5 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 the crude sample containing target protein and incubate at room temperature or desired temperature for 1-2 hours (Lower temperature require longer incubation time).
5. Extensively wash the beads with 5 bead volumes of PBS, pH 7.4, 0.5 M NaCl (or 1M NaCl ) until the absorbance of eluting at 280 nm approaches background level (OD 280 < 0.05).
6. Elute the target protein by appropriated methods such as low pH (2-4), high pH (10-12), high salt, high temperature, affinity elution or boiling in SDS-PAGE sample buffer.