rProtein A Magarose Beads

rProtein A Magarose Beads                

1. Product Description

Magarose Beads have superparamagnetism and rapid magnetic responsiveness. Peptides, proteins, antibodies, oligomeric nucleotide biological ligands can be  coupled to the surface of microspheres. Magarose Beads is an important carrier of the medical and molecular biology research tools.The particle size of Magarose Beads is about 30 to 100 µm and it is more suitable for biological examination and purification experiment.

rProtein A Magarose Beads is an affinity chromatography Magnetic beads designed for easy, one-step binding of classes, subclasses and fragments of immunoglobulins from biological fluids and from cell culture media. The recombinant protein A is coupled to Magnetic beads. Protein A, a bacterial cell wall protein isolated from Staphylococcus aureus, binds to mammalian IgGs mainly through Fc regions. Recombinant protein A contains five high affinity IgG binding domains with other non-essential domains removed to reduce nonspecific binding. Since only the Fc region is involved in binding, the Fab region is available for binding antigens.

Table 1. Characteristics of rProtein A Magarose Beads

Table 2. Relative binding strengths of antibodies from various species to protein G and protein A as measured in a competitive ELISA test.

++++=strong binding; ++= medium binding; —=weak binding or no binding

2. Purification Procedure

This protocol offers a general guideline for immunoprecipitation. Optimization may be required for each antibody and target antigen. The protocol uses 100 μl of rProtein A Magarose Beads, but this may be scaled up or down as required.

2.1 Buffer Preparation

Water and chemicals used for buffer preparation should be of high purity. It is recommended to filter the buffers by passing them through a 0.22 or 0.45 μm filter before use.

Binding/Wash Buffer: PBS, pH 7.4 (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4)

Acid Elution Buffer: 0.1 M glycine, pH 3.0

Neutralization Buffer: 1 M Tris, pH 8.5

2.2 Preparation of the Magnetic Beads

1) Completely resuspend the beads by shaking or vortexing the vial.

2) Transfer 500 μl rProtein A Magarose Beads (20% v/v) into a clean tube.

3) Place the tube on a magnetic separation rack to collect the beads. Remove and discard the supernatant.

4) Add 1 ml Binding/Wash Buffer to the tube and invert the tube several times to mix. Use the magnetic separation rack to collect the beads and discard the supernatant. Repeat this step twice.

2.3 Protein Purification 

1) Resuspend the beads in 100 μl Binding/Wash Buffer.

2) Add the sample to the tube and gently invert the tube to mix.

3) Incubate the tube at room temperature with mixing (on a shaker or rotator) for 30 min. It also can be done at 4℃ over night.

4) Use the magnetic separation rack to collect the beads and discard the supernatant. If necessary, keep the supernatant for analysis.

5) Add 1ml Binding/Wash Buffer to the tube and mix well, use the magnetic separation rack to collect the beads and discard the supernatant. Repeat the wash step three more times.

2.4 Elution of Target Protein

Add 300-500 μl Elution Buffer, mix well and incubation for 5-10 min (on a shaker or rotator). Use the magnetic separation rack to collect the beads and transfer the supernatant into a new tube. Repeat this step one more time.

Add 50 μl Neutralization Buffer to each 500 μl of eluate to neutralize the pH.

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