ANTI-PROTEIN C AFFINITY MATRIX

General description

Protein C is a Vitamin K-dependent plasma zymogen that is activated by proteolytic cleavage of the thrombin-thrombomodulin complex to form an anticoagulant enzyme. Anti-Protein C mouse monoclonal antibody (clone HPC4) binds specifically to an epitope sequence spanning the thrombin cleavage site of protein C and is immobilized. Anti-Protein C recognizes the 12-amino acid sequence (EDQVDPRLIDGK), which encodes residues 6 through 17 of the heavy chain of Protein C. The formation of the Anti-Protein C/protein C epitope complex is dependent on the presence of calcium ions. In the presence of Ca²⁺, the antibody binds with high affinity and specificity to this sequence in native human Protein C or in proteins tagged with this epitope. This efficient binding within the recombinant fusion protein occurs regardless of the site of incorporation of the epitope tag (i.e., N terminus, C terminus, or within the reading frame). This unique antibody is especially well suited for purification of recombinant fusion proteins tagged with the protein C epitope.

• Insertion of the protein C tag does not introduce a new metal-ion binding site. The antibody contains the Ca²⁺ binding site.
• Protein C tag can be integrated either at the N-terminus, C-terminus or internally without any change in antibody specificity.
• Rapid immunoaffinity purification under non-denaturing conditions using economical calcium chelating agent (e.g., EDTA) or alternatively a specific protein C-tag peptide.

Monoclonal mouse antibody Anti-Protein C (clone HPC4) is covalently coupled to agarose beads. In the coupling reaction 4mg of antibody is reacted per 1ml of beads.

Specificity

Anti-Protein C recognizes the 12-amino acid sequence EDQVDPRLIDGK, which encodes residues 6 to 17 of the heavy chain of protein C. In the presence of Ca², the antibody binds with high affinity and specificity to this sequence in native human protein C or in proteins tagged with this epitope. Efficient binding within the recombinant fusion protein occurs regardless of epitope position (N-terminal, C-terminal, or internal).

Application

Anti-Protein C Affinity Matrix is used for:

• Immunoprecipitation of Protein C-tagged proteins from mammalian, bacterial, and yeast cell extracts
• Affinity column purification of Protein C-tagged proteins from crude protein extracts

Following immunoprecipitation or purification, the tagged protein of interest may be analyzed by:

• Western blotting using the Anti-Protein C antibody
• Silver staining (or similar protein stain)

Features and Benefits

• use gentle elution conditions using calcium-chelating agents like EDTA.
• highly specific to EDQVDPRLIDGK, derived from protein C.
• binding of Anti-Protein C to protein C-epitop is dependent on the presence of calcium ions.
• suitable for purification of proteins containing protein C as N-terminal, C-terminal or internal fusion.
• applicable with crued cell extracts from mammalian, bacterial, and yeast expression systems.

Contents
1. The antibody is covalently coupled to agarose beads and supplied as a 2ml slurry containing 1ml beads and 1ml buffer.
2. 4mg of antibody is reacted per ml of beads in the coupling reaction.
3. A plastic column with top and bottom caps is included.

Packaging

1 kit containing settled resin and column

Quality

Each lot of Anti-Protein C Affinity Matrix is tested for its ability to purify a Protein C-tagged protein expressed in transformed bacteria from crude bacterial extract. The antibody affinity column is used in combination with western blot and/or silver stain analysis.

Physical form

1 ml settled resin of Anti-Protein C Affinity Matrix in 20 mM Tris, 0.1 M NaCl, 1 mM CaCl₂, and 0.09% sodium azide (w/v); 2 ml suspension equals to 1 ml bed volume. One plastic column with top and bottom caps is included

Analysis Note

K_D = 10^–9 M Binding Capacity is 2 to 10 nmol/ml affinity matrix. Yield of 10.5 nmol purified protein/ml affinity matrix was determined using a whole-cell bacterial extract containing protein C-tagged β-galactosidase.

Other Notes

For life science research only. Not for use in diagnostic procedures.