The NHS-activated magnetic product consists of super-paramagnetic beads which are functionalized with amino-reactive groups. For covalent coupling of molecules with primary amino groups such as peptides, proteins, antibodies and aptamers. Subsequently, target proteins are affinity purified using magnetic separation technology.
The magnetic beads show superior magnetic behavior and are easily attracted to external magnets allowing separation within seconds. The matrix enables minimal nonspecific binding of proteins etc. due to its hydrophilic nature. The beads are black and easily observed by the eye and dense making them easy to spot and collect. The beads do not aggregate and are easily re-suspended in most buffers.
The coupling capacity is general 1-10 mg protein per ml beads.
The number of beads needed can easily be scaled up or down to match antibody concentration and sample volumes. The beads are suitable for separations from ul to 500 ml scale using appropriate magnetic separators.
||Pre-activated and ready to use of 30mg dry beads.
The magnet is not provided.
||Amino groups multi-point attachment
||100 mM sodium phosphate pH 7.4, 150mM
||1-10 mg protein or 1 mg peptide per 30mg beads.
Coupling capacity was determined by incubating 30mg beads with human lgG (1 mg/ml in 1ml PBS) for 60 minutes at room temperature.
||-20°C, free of moisture upon receipt
| Stable for at least 4 months|
Coupling buffer (For coupling of antibodies, proteins or aptamers to beads and for washing out unbound molecules from beads.): 100 mM sodium phosphate pH 7.4, 150 mM NaCl
Washing buffer: 0.05 M Tris-HCl, 0.5M Nacl, pH 8.0
Blocking buffer (To block remaining reactive groups on the
beads.): 1 M Ethanol amine, pH 9.0
- There are 30mg Magnetic beads in the centrifuge tube.
- Dissolve 0.5-1mg protein/peptide in 1ml coupling buffer.
Coupling efficiencies to NHS-activated magnetic beads vary from ligand to ligand. The user should empirically optimize the concentration of the ligand. 0.5-10 mg/ml is recommended for protein conjugation. For small peptides, the concentration of ligand should be at least 200 um ligand per ml.
- Add the protein solution to the beads. Resuspend the magnetic beads and mix very well by pipetting or vortexing. Incubate the reaction with continuous rotation at room temperature for 4-6 hours or overnight.
- Place the tube on the magnetic separator for 1-3minutes. Remove the supernatant while the tube remains on the separator. Remove the tube from the separator and resuspend the beads with 1 ml wash buffer by vortex for 30 seconds. Place the tube on the magnetic separator for 1-3 minutes. Remove the supernatant while the tube remains on the separator.
- Wash beads 3-4 times with 1 ml wash buffer (or 1M NaCl) as described at step 4.
- Add 0.5-1ml blocking buffer (Beads can also be blocked by PBS, pH7.4, 0.1% BSA) to the beads and incubate at room temperature for 1 hour or at 4 °C overnight.
- Wash the beads with 1 ml of cold Wash buffer 3 times as described at step 4.
- Resuspend the beads in PBS buffer, pH7.4, 0.1% BSA and 0.1% azide (w/v) to desired concentration and store at 4°C until use. Do not freeze.
General Affinity Purification Protocol
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.
- 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 a 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.
- 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.
- Repeat step 2 two times.
- 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).
- 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).
- 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.
The product is not fully tested and is not intended for human use. For
in-vitro and research use only.