Protein G agarose Resin 6FF for Antibody Purification

Product

Protein G agarose Resin 6FF for Antibody Purification

Catalog number :P2709

Protein G Agarose Resin 6FF is a high-performance affinity resin for antibody purification that is available as bottled agarose beads, pre-packed columns, and complete IgG purification kits.

Protein G Agarose Resin 6FF consists of purified recombinant Protein G that has been covalently immobilized at high density onto high-quality crosslinked 6% beaded agarose. Among the many available varieties of immobilized Protein G affinity resins, this one provides the most versatile combination of chromatographic features for high yield and high purity purification of whole IgG from mammalian serum samples. The agarose beads have physical and chemical properties suitable for many affinity purification systems. Accordingly, Protein G Agarose Resin 6FF is offered in several convenient package formats, including bottled resin slurries, three sizes of spin columns, complete purification kits, two sizes of FPLC-ready chromatography cartridges.

Protein G is a bacterial cell wall protein isolated from group G Streptococci. DNA sequencing of native Protein G identifies two IgG-binding domains and sites for albumin and cell surface binding. The albumin and cell surface binding domains have been eliminated from Recombinant Protein G to reduce nonspecific binding and, therefore, can be used to separate IgG from crude samples. Optimal binding occurs at pH 5, although binding is also effective at pH 7.0 to 7.2.

Features of Recombinant Protein G:

• Contains two Fc-binding domains per protein

• Better than Protein A for mouse (including IgG1), human (including IgG3) and rat, goat and cow antibodies

• Poorer than Protein A for guinea pig, pig, dog and cat

• Does not bind human IgM, IgD or IgA

Overview

Description: Protein G Resin for Antibody Purification

Tested applications: Antibody Purification

Product Picture

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

Form: 50% slurry

Storage instruction: Store Protein G Resin in 1*PBS, containing 20% Ethanol at 2°C to 8°C. Do not freeze.

Beads Diameter: 90 μm

Binding Capacity: >40 mg human or mouse IgG/mL settled resin

Ligand: Recombinant protein G produced in E.coli

Matrix: Highly cross-linked 6% agarose beads

Maximum pressure: 0.3 MPa (3 bar)

Chemical stability: All commonly used aqueous buffers, including 6 M guanidine-HCl and 8 M urea.

Flow rate: Gravity flow, 100 - 300 cm/hour

pH stability: Long term: pH3 - 9;

Short term: pH2 - 10

Storage buffer: 1x PBS containing 20% ethanol

Applications

Application Image

Work Flow Image

Application Figure 1: High purity and high capacity of rProtein G 6FF resin.

5 mL mouse ascites fluid was loaded on the gravity flow column containing 0.5 mL settled rProtein G 6 FF resin. Bound immunoglobulin were eluted with 100 mM Glycine, pH 3.0. Fractions were analyzed by SDS-PAGE.

Lanes: (1) Raw ascites fluid; (2) Flow-through; (3) Elution 1; (4) Elution 2.

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

Protocols: Antibody Purification Protocol (Gravity-flow column)

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

Additional Solutions and Materials Required

• Gravity-flow column of 10-15 mL bed volume

• 15mL collection tubes

• Binding Buffer: 100mM phosphate, 150mM sodium chloride; pH 7.2 when dissolved in 500mL of ultrapure water

• IgG Elution Buffer: 0.1M glycine, pH 2-3

• Neutralization Buffer: 12mL, 1M Tris•HCl, pH 8.5

• Storage solution: 0.02% sodium azide in phosphate-buffered saline (PBS, pH 7.4)

Antibody Purification Procedure

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

2. Dilute sample at least 1:1 with Binding Buffer before purification using the Protein G 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 G 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.