The Magnetic Agarose consists of super-paramagnetic agarose beads which are functionalized with amino-reactive groups. For covalent coupling of molecules with primary amino groups or thiol such as proteins, antibodies and aptamers. Subsequently, target proteins are affinity purified using magnetic separation technology.
The magnetic agarose beads show superior magnetic behavior and are easily attracted to external magnets allowing separation within seconds. The agarose 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 0.5-1 mg human lgG per ml of 10% bead suspension.
The quantity 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.
||1 mL Amino Group Conjugation Magnetic Agarose 10%
suspension in 15 mM sodium phosphate pH 7.4, 150 mM NaCI, including 20% ethanol.
1 piece of Neodymium cube magnet (12mm) suitable for separations in 0.5-5 ml volumes.
||Amino groups multi-point attachment
||45 μm - 165 μm
|15mM sodium phosphate pH 7.4, 150mM
||0.5-1mg protein or peptide per ml bead solution.
Coupling capacity was determined by incubating 1 ml 10% Agarose with human lgG (1 mg/ml in 1ml PBS) for 60 minutes at room temperature.
||2-8°C in 20% ethanol
| 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.): 15 mM sodium phosphate pH 7.4, 150 mM NaCl
Washing buffer: 15 mM sodium phosphate pH 7.4, 150 mM NaCl
Blocking buffer (To block remaining reactive groups on the
beads.): Ethanol amine (50% v/v in binding buffer)
Binding buffer (To bind your target protein to the functionalized beads.): 15 mM sodium phosphate pH 7.4, 150mM NaCl
Elution buffer (To elute your target protein from the functionalized beads.): 60 mM citric acid pH 2.2 to 3.0 or 50 mM glycine-HCI pH 2.2 to 3.0
Storage buffer (For storage of beads.): 15 mM sodium phosphate pH 7.4, 150mM NaCI, including 20% ethanol
Protocol to Couple amino-containing peptide or proteins to Magnetic Agarose
- Equilibration for coupling: Dispense the affinity magnetic beads in a test tube. 1000μl of
10% well suspended (100μl bead volume) beads are suitable
to couple 0.5-1mg protein.
Remove the storage solution by magnetic separation.
Re-suspend the magnetic beads in 10 bead volumes (BV) of
Remove the liquid by magnetic separation.
Re-suspend the magnetic beads in binding buffer.
- Coupling: Dissolve amino-containing ligand (protein, peptide etc.) at a
concentration of 1-2 mg/ml in binding buffer.
Add the amino-containing ligand to the magnetic bead
suspension, total volume of approximately 10 BV, and allow
coupling for 1 hour at room temperature on vortex/mixing. Coupling efficiency of proteins and peptides could be
determined by A280 measurements of the ligand solution
before and after coupling to beads.
- Washing: Remove coupling solution from beads by magnetic
Wash out unbound ligand with 10 BV binding buffer.
Repeat washing step twice.
- Blocking: Remaining reactive groups on beads are blocked with
Add typically 80μl ethanol amine (50% v/v in binding buffer)
to 1ml bead solution.
Allow blocking for 45 minutes at room temperature on
vortex/mixing. Wash with 10 BV binding buffer and repeat washing step
Resuspend beads in 10BV binding buffer, 10% medium, if
beads are used within the following days.
Beads ready for use.
For longer storage resuspend beads in 20% ethanol solution.
- Equilibration for binding: Remove the storage solution by magnetic separation. Re-suspend the magnetic beads in l 0 BV of binding buffer. Remove the liquid by magnetic separation.
- Binding of target protein: Conditions for this and following steps depend on the bound ligand. Literature references might be consulted. Add target protein sample (diluted in e.g. binding buffer) and allow coupling for l hour at room temperature on vortex/mixing. Remove and collect the non-bound fraction by magnetic separation.
- Washing: Remove sample solution from beads by magnetic separation.
Wash out unbound sample with 10 BV binding buffer. Repeat washing step twice.
- Elution of target protein: Add 3-l0 BV of elution buffer and mix the solution at room temperature by vortexing or with gentle manual inversion of the test tube. After 15 minutes remove and collect the elution fraction, which
contains the main part of the target protein. Repeat the elution step if required, e.g. if low amount of target protein is obtained in the first elution step.
- Instead of using a magnetic separator in order to separate
beads from solution in the washing steps, beads can also be
washed on a sintered filter funnel.
- 1000μl of 10% well suspended (100μl bead volume) beads are suitable to couple 0.5-1mg protein or peptide.
- Beads caught in the lid or on the walls of the reaction
vial/test tube can be removed by pipetting.
- If the magnetic separation of the magnetic beads are
insufficient or beads are lost when washing use less amounts
of beads or a stronger magnetic separator.
The product is not fully tested and is not intended for human use. For
in-vitro and research use only.