Smart-antibody™ Protein A Antibody Purification Kit

Overview

Description

Protein A Antibody Purification Kit (Agarose Beads)

Tested applications

Antibody Purification ( by use protein A agarose beads)

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Specificity

Protein A and G are popular choices for antibody purification, because they are both stable and target selective. IgG class antibodies from multiple species bind to protein A and/or G, allowing antibody to be captured on protein-bound beads. Protein A and G bind IgG subtypes with varying affinities, determined by species and the properties of the heavy chain. This chart that reports the affinity of protein A and G for various species immunoglobulin subtypes.

Properties

Storage instruction

2 - 8°C, do not freeze

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Binding Capacity

>40 mg human IgG/mL settled resin

Applications

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Purification of rabbit IgG using Protein A resin 6FF.

High purity and high capacity of Protein A resin 6FF. 

 

5 mL rabbit serum was loaded on the column containing 0.5 mL settled Protein A resin 6FF. Bound immunoglobulin species were eluted with 100 mM Glycine, pH 3.0. Fractions were analyzed by SDS-PAGE.

 

Lanes: (1) Raw rabbit serum; (2) Flow-through; (3) Elution.

 

Note that, heavy and light chains of IgG were mostly absent from the flow-through, indicating that most immunoglobulins were bound to the column.

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Protocols

Antibody Purification Protocol (Gravity-flow column)

Note: The following protocol is for using a gravity-flow column packed with 1mL of Protein A agarose beads (i.e., 2mL of the 50% beads slurry). When using columns containing other resin volumes, reagent amounts must be adjusted accordingly. 

 

Antibody Purification Procedure

1. Equilibrate column, protein A agarose beads and buffers to room temperature.

 

2. Dilute sample at least 1:1 with Binding Buffer before purification using the Protein A beads Column to maintain the proper ionic strength and pH for optimal binding.

Note: Plasma may become hazy upon dilution with the Binding Buffer because of lipoprotein precipitation. Centrifuge the diluted sample at 10,000 × g for 20 minutes and apply the supernatant to the equilibrated Protein A agarose beads.

 

3. Gently pack the column with 2mL of resin slurry and allow storage solution to drain.

 

4. Equilibrate column by adding 5mL of Binding Buffer and allow the solution to drain through the column.

 

5. Apply the diluted sample to the column. For best results, add a sample volume that is less than 80% of the column’s antibody-binding capacity. Collect the flow-through.

Note: If the sample contains more IgG than can bind to the column, the flow-through will contain the excess antibody. By saving the flow-through, nonbound antibody can be recovered and analyzed.

 

6. Wash column with 15mL of Binding Buffer.

Note: If desired, verify that all nonbound proteins are removed from the column by collecting separate 2mL fractions as the column drains. Measure the absorbance of each fraction at 280nm. The last fractions should have an absorbance similar to the Binding Buffer.

 

7. Add 100μL Neutralization Buffer to collection tubes. Elute antibodies with 5mL of Elution Buffer, collecting 0.5-1mL fractions in the neutralization buffer-containing collection tubes. Monitor the elution by measuring the absorbance at 280nm or by protein assay such as BCA Protein Assay.

 

8. Pool the eluted IgG fractions that contain the highest absorbance. The purified antibodies may be used directly for SDS-PAGE, or the buffer may be exchanged by dialysis or de salting column to one that is compatible with the specific downstream application.

 

9. Regenerate column by adding 15mL of Elution Buffer and allow solution to flow through the column. Columns may be regenerated at least 10 times without significant loss of binding capacity.

 

10. Store column by adding 5 ml of storage solution. When approximately 3mL remain in the column, cap bottom and secure top cap. Store column at 4°C.