Pierce™ Protein L Agarose (Thermo Scientific™)

The Thermo Scientific NAb Protein L Spin Columns are convenient for rapid, small-scale affinity purification of antibodies from a variety of sample types. Each column containing 5 mL of the immobilized protein resin enables quick purification of 5-65 mg of IgG from 2-10 mL of serum or other sample. The actual amount of IgG purified varies depending upon the sample type and the specific spin column used.

Pierce Protein L Agarose consists of recombinant Protein L that has been covalently immobilized onto high-quality crosslinked 6% beaded agarose (CL-6B). This particular variety of the resin provides the most versatile combination of chromatographic features for high yield and high purity purification of target immunoglobulins. The agarose beads have physical and chemical properties suitable for many affinity purification systems.

Features of Pierce Protein L Agarose:

• Protein L – immobilized Protein L is ideal for selective purification of human and mouse antibodies that have kappa light chains
• Agarose resin – support is crosslinked 6% beaded agarose (CL-6B), the most popular resin for protein affinity purification methods
• Inert and stable – superior manufacturing method immobilizes Protein L by charge-free, leach-resistant covalent bonds, resulting in low nonspecific binding and enabling multiple uses without decline in yield
• Standard capacity – Pierce Protein L Agarose has a normal load of immobilized Protein L, providing a binding capacity of 5 to 10 mg human IgG/mL resin

Applications:
• Purify monoclonal and polyclonal antibodies that bind poorly to Protein A or Protein G agarose
• Purify monoclonal antibodies from culture supernatants supplemented with bovine serum
• Protein L immunoprecipitation and co-immunoprecipitation assays
• Purify ScFv and Fab fragments containing kappa light chains

Protein L is an immunoglobulin-binding protein (36kDa) that originates from the bacteria Peptostreptococcus magnus, but is now produced recombinantly in E. coli. Unlike Protein A and Protein G, which bind primarily through Fc regions (i.e., heavy chain) of immunoglobilins, Protein L binds Igs through interactions with the light chains. Because no part of the heavy chain is involved in the binding interaction, Protein L binds a wider range of Ig classes than Protein A or G. Protein L binds to representatives of all classes of Ig, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (ScFv) and Fab fragments also bind to Protein L.

Despite this wide-ranging binding capability with respect to Ig classes, Protein L is not a universal immunoglobilin-binding protein. Binding of Protein L to immunoglobulins is restricted to those containing kappa light chains (i.e., kappa chain of the VL domain). In humans and mice, kappa light chains predominate. The remaining immunoglobulins have lambda light chains. Furthermore, Protein L is effective in binding only certain subtypes of kappa light chains. For example, it binds human VkI, VkIII and VkIV subtypes but does not bind the VkII subtype. Binding of mouse immunoglobulins is restricted to those having VkI light chains.

Given these specific requirements for effective binding, the main application for immobilized Protein L is purification of monoclonal antibodies from ascites or culture supernatant that are known to have the kappa light chain. Protein L is extremely useful for purification of VLk-containing monoclonal antibodies from culture supernatant because it does not bind bovine immunoglobilins, which are present in the media serum supplement. Also, Protein L does not interfere with the antigen-binding site of the antibody, making it useful for immunoprecipitation assays, even using IgM.

Pierce Protein L Agarose is prepared using Thermo Scientific AminoLink Coupling Chemistry, which provides several advantages compared to traditional methods of ligand immobilization. AminoLink Immobilization results in conjugation between sugar monomers of the agarose beads and native lysine residues on the Protein A via simple amide bonds. Unlike typical cyanogen bromide (CNBr) immobilization, the AminoLink Method does not introduce any novel chemical groups that could cause undesired nonspecific binding and produces a stable, essentially irreversible bond. The result is a high-binding-capacity resin that retains functional immobilized Protein L through multiple rounds of antibody purification.

Pierce Protein L Agarose is a versatile affinity resin for purification of mammalian IgG isotypes that contain specific kappa light chains. Protein L is especially suited for isolating mouse IgG VkI and human IgG VkI, VkIII and VkIV monoclonal antibodies.

Properties of crosslinked 6% beaded agarose (CL-6B):
• Support pH Stability: 2 to 14 (short term); 3 to 13 (long term)
• Average Particle Size: 45 to 165 microns
• Exclusion Limit: 10,000 to 4,000,000 daltons
• Maximum Volumetric Flow Rate: approx. 1 mL/minute (for 1 cm diameter column)
• Maximum Linear Velocity: 30 cm per hour
• Maximum Pressure: less than 25psi (1.5 bar), defined as the maximum pressure drop across a column that the resin can withstand (Note: The indicated gauge pressure of a liquid chromatography apparatus may be measuring the total system pressure rather than the pressure drop across the column.)

More Product Data
• Using antibody-binding proteins for antibody purification

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