Protein L Agarose

Protein L Agarose

Introduction:

Protein L was isolated from the surface of bacterial species Peptostreptococcus magnus and was found to bind Ig(IgG,IgM,IgA,IgE and IgD) through L chain interaction. The recombinant protein contains four immunoglobulin (Ig) binding domains (Bdomains) of the native protein. Recombinant Protein L Protein, fused with the polyhistidine tag at N-terminus and a single cysteine at C-terminus. DTT-reduced protein migrates as a 36 to 38 kDa polypeptide.

Protein L agarose is prepared by coupling Protein L to agarose to stabilize the binding characteristics of Protein L.

Amino Acid Sequence:
Approximately 50.5 kDa. The recombinant Protein A/G consists of 7 IgG-binding domains EDABC-C2C3, which corresponds to the Protein A and G domains that are included in the recombinant sequence. The Protein A portion is from Staphylococcus aureus segments E, D, A, B and C. The Protein G portion is from Streptococcus segments C2 and C3. The binding capacity of recombinant Protein A/G is broader than either Protein A or Protein G alone.

Formulation: 4 ml Crosslinked 6% beaded agarose supplied as a 50% slurry in PBS, 0.02% sodium azide.

Binding capacity:  >5mg human IgG/mL of resin

Specificity:
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., k chain of the VL domain). Besides antibody, protein L is also suitable for binding of a wide range of antibody fragments such as Fabs, single-chain variable fragments (scFv), and domain antibodies.

Usage:
This product is for research use only. It is not approved for use in humans, animals, or in vitro diagnostic procdures.

Gravity-flow Column Procedure for Antibody Purification for Protein L Agarose (also for protein A, G and A/G)

A. Buffer composition

• Binding Buffer: 0.1M phosphate, 0.15M sodium chloride; pH 7.2

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

• Neutralization Buffer: 1M Tris, (pH 8.5)

B. Immunoglobulin Purification Procedure (a column for 2 mL of protein L Agarose)

1.     Equilibrate the Protein L agarose and reagents to room temperature before use.

2.     Carefully pack the Protein L resin slurry (4mL) into the column. Equilibrate the Protein L column by adding 10mL of the Binding Buffer and allowing the solution to drain through the column.

3.     Dilute sample at least 1:1 with Binding Buffer before applying onto the Protein L column to maintain optimal ionic strength and pH for binding. Note: If plasma is used, centrifuge the diluted sample at 10,000 × g for 20 minutes and apply the supernatant to the equilibrated Protein L column.

4.     Apply up to 4mL of the diluted sample to the column and allow it to flow completely into the resin. The column will stop flowing automatically when the liquid level reaches the top disc. Note: By collecting the flow-through, non-bound antibody can be recovered and examined by antibody-specific assays.

5.     Wash the Protein L column with 10-15mL of Binding Buffer. If necessary, measure the A280nm of elution, the last fractions should have absorbances similar to Binding Buffer alone.

6.     Elute antibodies with 6-10mL of Elution Buffer and collect 0.5-1mL fractions. Immediately adjust eluted fractions to physiologic pH by adding 100μL of the Neutralization Buffer to 1mL of eluate.

7.     Pool the eluted Ig fractions that contain the highest absorbance by measure the A280nm.

8.     Regenerate column by washing with 12mL of Elution Buffer.

9.     For storage, wash column with 5mL of water containing 0.02% sodium azide. When approximately 3mL of solution remains, replace the bottom cap followed by the top cap on the column. Columns may be regenerated a minimum of 10 times without significant loss of binding capacity.