Peptide Conjugation Magnetic Agarose, 10% bead suspension

Catalog Number:
LT13512
Packing Details:
2x1 mL Protein A Magnetic Agarose 10% suspension
Binding Capacity:
> 3 mg human IgG / ml 10% bead suspension
> 30 mg human IgG / ml settled beads
Particle size:
45 μm - 165 μm
Recovery:
> 85%
IgG binding conditions:
15 mM phosphate pH 7.4,
150 mM NaCl
IgG elution conditions:
60 mM citrate pH 3.0 or
50 mM glycine pH 2.5
Storage:
2-8°C in 20% ethanol
Shelf Life:
Stable for at least 4 months
Description:

Principle of action

  • Protein A Magnetic Agarose beads are added to the sample containing antibodies.
  • The beads will capture the antibodies during a short incubation time and are then separated from the sample using an external magnet.
  • The bead/antibody complex is washed to remove unbound impurities.
  • The purified antibodies are dissociated from the beads by the addition of a low pH elution buffer.

Material Supplied

  • Vials with Protein A Magnetic Agarose supplied as a 10% bead suspension in 20% ethanol.
  • 1 piece of Neodymium cube magnet (12mm) suitable for separations in 0.5-10ml vials.

Additional materials needed

  • Binding/Washing buffer - For coupling of antibodies to beads and for washing, use PBS (15mM phosphate, 150mM NaCl, pH 7.4)
  • Elution buffer - For release of antibodies from beads, use 60mM Citrate, pH 3.0 or 50mM Glycine, pH 2.5
  • Neutralization buffer - To neutralize eluted antibodies use, 1M Tris-HCl, pH 7.5

Antibody purification protocol

Homogenize the bead suspension by using a vortex or by manual inversion before dispensing the magnetic bead suspension into a test tube. Use a magnetic separator in order to attract the magnetic agarose beads to the wall of the test tube before each liquid removal step. Be careful when removing liquid with the pipet tip so as not to remove magnetic agarose beads, leading to sample loss. Re-suspend the agarose beads in absence of the magnetic separator by vortexing or by manual inversion, after each liquid addition step.

1. Bead Pre-treatment

  • Dispense the affinity magnetic beads in a test tube. Use 500 μl of well suspended 10% Protein A Magnetic Agarose (bead volume (BV): 50 μl) for each 1-2 mg antibodies (Ab) to be purified.
  • Remove the storage solution by magnetic separation.
  • Re-suspend the magnetic beads in 10-20 bead volumes (BV) of binding buffer.
  • Remove the liquid by magnetic separation.
  • Re-suspend the magnetic beads in 5 BV binding buffer.

2. Sample Pre-treatment

  • Adjust the pH of the sample by diluting the sample with binding buffer. The sample pH should be equal to the binding buffer pH. The sample volume should at least be 5 BV.

3. Sample Application

  • Add the sample to the equilibrated magnetic bead suspension.
  • Mix the solution during the binding at room temperature by vortexing or with gentle manual inversion of the test tube. IgG will bind to the Protein A Magnetic Agarose beads.
  • Remove the liquid after 15-60 minutes by magnetic separation. It is recommended to optimize the coupling time of antibodies to beads depending on sample source and antibody concentration.

4. Washing out Unbound Sample

  • Add 10-20 BV of binding buffer and re-suspend the magnetic beads.
  • Remove the liquid by magnetic separation.
  • Repeat the washing step twice.

5. Elution

  • Add 3-10 BV of elution buffer to the washed magnetic beads.
  • 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 purified antibody. If necessary, adjust the pH to neutral to preserve activity of the eluted antibodies.
  • Repeat the elution step if required, e.g. if low amount of antibody is obtained in the first elution step.

6. Regeneration of the Magnetic Beads

  • Wash the magnetic beads with 10 BV elution buffer.
  • Remove the liquid by magnetic separation.
  • Wash the magnetic beads two times with 10 BV binding buffer.
  • Remove the liquid by magnetic separation.
  • Re-suspend the magnetic beads in 10 BV storage solution (10% bead suspension) and store at 2-8°C.

7. Practical notes

  • Use approximately 500 μl 10 % bead suspension (50μl settled beads) to purify 1-2 mg Ab.
  • Beads caught in the lid or on the walls of the reaction vial can be removed by pipetting.
  • If separation of the magnetic beads are insufficient or beads are lost when washing, use less amounts of beads or a stronger magnetic separator.
  • If low amount of antibody is recovered increase the amount of magnetic beads or increase the time of coupling. It is recommended to optimize the coupling time of antibodies to beads depending on sample source and antibody concentration.
  • If antibody is degraded in the elution buffer, add neutralization buffer to neutralize the pH.
  • If beads are aggregated between uses, vortex the bead suspension vigorously before next use.
  • When reusing beads, it is recommended to purify the same antibody in order to avoid any cross-contamination between the purification runs.
Typical binding and elution conditions with protein A Agarose

Species

Antibody Class

Protein A binding

pH value of binding buffer

pH value of elution buffer

Human

IgG1

++

6.0~7.0

3.5~4.5

IgG2

++

6.0~7.0

3.5~4.5

IgG3

-

8.0~9.0

<7.0

IgG4

++

7.0~8.0

2.5~4.5

Cow

IgG2

++

 

2

Goat

IgG2

+

 

5.8

Mouse

IgG1

+

8.0~9.0

5.5~7.5

IgG2a

+

7.0~8.0

4.5~5.5

IgG2b

+

7.0

3.5~4.5

IgG3

+

7.0

4.0~7.0

Rat

IgG1

+

>9.0

7.0~8.0

IgG2a

-

>9.0

<8.0

IgG2b

-

>9.0

<8.0

IgG3

+

8.0~9.0

3.0~4.0

Rabbit

no distinction

++++

7.4

3.5~4.5

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