Amine-Terminated Peptide Conjugation Magnetic Beads

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Click here for the Amine-Activated Magnetic Beads.

Click here for the Thiol-Activated Peptide Conjugation Magnetic Beads.

Faster Magnetic Response; Simpler Protocols;

LifeTein’s unique coupling chemistry enables fast and efficient conjugation of amino (-NH2) containing proteins and peptides, to the solid core with iron oxide clusters. All steps in the coupling are performed at physiological conditions and at room temperature with high yields and short reaction times. The magnetic product consists of nano-superparamagnetic beads, which are functionalized with high-density maleimide groups. The beads couple thiol-containing ligands such as peptides, proteins, antibodies, and aptamers. Subsequently, target proteins are easily separated from the solution using a magnet for downstream experiments.

The Amine-Terminated Magnetic Beads are silica-based superparamagnetic beads coated with a high density of primary amine functional groups on the surface. The beads are used to conjugate primary amine or carboxy-containing ligands covalently.

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 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 generally 1-10 mg protein or 0.1-1mg peptide/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 the ul to 500 ml scale using appropriate magnetic separators.

Magnetic bead
Catalog Number:
LT16323-2
Packing Details:
30mg, in 20% ethanol

The magnetic separator is not provided.

Binding Capacity:
1-10 mg protein or 0.1-1mg peptide/30mg magnetic beads.
Particle size:
5um diameter
Coupling conditions:
10 mM pyridine
Coupling capacity:
1-10 mg protein or 1 mg peptide/30mg beads.

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

Storage:
Product shipped at room temp. Upon receipt, please store at 2-8°C.
Shelf Life:
Stable for at least two years
Recommended Buffers:

Solvent: 20% Ethanol (Concentration: 30mg/ml)

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

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

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.

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 final volume to 100 ml with ddH2O

Description:

Magnetic Beads Preparation

1.Suspend the magnetic beads with 20% Ethanol (Concentration: 30mg/ml).

2.Transfer and wash the beads by adding 1 ml coupling buffer and vortexing for 1-2 minutes.

3. Use a magnetic separator to separate and wash the beads 2-3 times with the coupling buffer.

4. Resuspend the magnetic beads by adding 20 ml of 5% Glutaraldehyde and shake vigorously. Leave at room temperature for 3 hr with gentle rotation.

5. Wash beads three times with 30ml coupling buffer as described to remove unreacted glutaraldehyde.

Coupling of Protein

Note:

For some expensive proteins, such as monoclonal antibodies, the supplied concentration cannot reach the required 2.5-10mg/ml. To ensure highly efficient coupling, the BSA should be added to the protein solution to bring protein concentration to the required level.

1. Prepare protein solution by adding 5-20 mg protein into 10 ml coupling buffer and mix very well.

2. Add the protein solution into the tube containing activated beads 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 40ml of reaction stop buffer into the tube. Shake vigorously to suspend the beads. Gently shake for 30 min at room temperature.

5. Wash the beads with 30 ml storage buffer three times.

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

General Affinity Purification Protocol

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 a titration be performed to optimize the quantity of beads used for each application based on the amount of the target protein in a crude sample. Too many magnetic beads used will cause higher backgrounds, while too few beads used will cause lower yields. Each mg of conjugated magnetic beads normally binds to 1-20 µg target protein.

2. Remove the tube and resuspend the beads with a 5-bed 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 a crude sample containing target protein and incubate at room temperature or desired temperature for 1-2 hours (Lower temperature requires longer incubation time).

5. Extensively wash the beads with 5 bed bead volumes of PBS buffer or 1M NaCl until the absorbance of elute at 280 nm approaches the background level (OD 280 < 0.05).

6. Elute the target protein by appropriate methods such as low pH (2-4), high pH (10-12), high salt, high temperature, affinity elution, or boiling in SDS-PAGE loading buffer.


The product is not thoroughly tested and is not intended for human use. For in-vitro and research use only.
  • 6 Units in Stock

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