Thiol-Activated Peptide Conjugation Magnetic Beads

Magnetic bead peptide pulldown

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The thiol-activated magnetic product consists of super-paramagnetic beads which are functionalized with high-density thiol functional (2-pyridyl disulfide) groups. The beads are used to reversible couple thiol-containing ligands 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 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 ul to 500 ml scale using appropriate magnetic separators.

Magnetic bead
Catalog Number:
LT16323
Packing Details:
Pre-activated and ready to use of 30mg dry beads.

The magnet is not provided.

Binding Capacity:
Thiol functional groups (2-pyridyl disulphide) on the surface
Particle size:
5um diameter
Coupling conditions:
0.1 M sodium phosphate, pH 7.0 , 5mM EDTA
Coupling capacity:
1-10 mg protein or 1 mg peptide/ml beads.

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

Storage:
-20°C, free of moisture upon receipt
Shelf Life:
Stable for at least 4 months
Recommended Buffers:

Coupling Buffer : 0.1 M sodium phosphate, pH 7.0 , 5mM EDTA

L-Cysteine•HCl

TCEP (tris(2-carboxyethyl)phosphine)

Washing Buffer: 1 M NaCl, 0.05% NaN3

Description:

Sample Preparation

1. Dissolve 1-10mg protein/peptide in 1ml coupling buffer.

2. If samples have already suspended in other buffer, dilute samples with equal volume of coupling buffer.

Bead activation:

Weight, suspend the magnetic beads with 20% Ethanol (Concentration: 30mg/ml), disperse the beads by vigorously vortexing and store at 4?C.

1. Transfer 30 mg Magnetic beads to a centrifuge tube. Resuspend the beads by adding 1 ml coupling buffer and mix the beads by vigorous vortexing for 1-2 minutes.

2. Place the tube on the magnetic separator for 1-3 minutes. Remove the supernatant while the tube remains on the separator. Remove the tube from the separator and resuspend the beads with 1 ml coupling buffer by vortex for 30 seconds.

3. Repeat step-2 once.

Protein Coupling

1. Add protein sample to the washed magnetic beads and incubate for 60 minutes at room temperature with gentle rotation.

2. Washed the magnetic beads with 1ml Coupling buffer for four times as described.

3. Block the excess active groups on the beads by suspending the beads in 1ml Coupling buffer containing 8mg L-Cysteine•HCl and incubate 30-60 minutes at room temperature with gentle rotation.

4. Wash the beads with 1ml Washing buffer four times as described .

5. Resuspend the beads in PBS buffer containing 0.05% sodium azide and store at 4C.

 

General Affinity Purification Protocol
1. Transfer 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.

2. Remove the tube and resuspend the beads with 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 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 bed bead volumes of PBS buffer or 1M NaCl until the absorbance of elute 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 loading buffer.

Release the thiol-containing ligand from magnetic beads

1. Resuspend the magnetic beads with 0.1 M DTT (dithiothreitol) or Mercaptoethanol solution and incubate at room temperature for 30 minutes with gentle rotation.

2. Place the tube on the magnetic separator for 1-3 minutes. Remove the supernatant containing the released ligand to a new centrifuge tube while the tube remains on the separator.

3. Perform buffer change by gel filtration or dialysis to dissolve the ligand into desired buffer.


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

$480.00  $320.00
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