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.

Application

Anti-Protein C Affinity Matrix is used for: Immunoprecipitation of Protein C-tagged proteins from mammalian, bacterial, and yeast cell extractsAffinity column purification of Protein C-tagged proteins from crude protein extractsFollowing immunoprecipitation or purification, the tagged protein of interest may be analyzed by: Western blotting using the Anti-Protein C antibodySilver 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.Contents1. 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.

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.

Other Notes

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

Packaging

1 kit containing settled resin and column

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

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.

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).