murine MAb against Gla (matrix bound) residues

Description
REF ADG3570Mx is a monoclonal antibody (REF 3570) directed against gamma-carboxyglutamyl (Gla) residues found in various proteins of human and other origin, and snake venoms, bound to SEPHAROSE™ CL-4B. It is intended for use in the affinity purification of Gla-containing proteins such as prothrombin, factor VII/VIIa, factor IX/IXa, factor X/Xa, Protein C, Protein S and growth arrest-specific protein-6 (Gas6).

Properties
The antibody is bound to the sepharose at a concentration of 5 mg of antibody/mL of settled gel.

Purification Protocol
Using the following buffers, a suggested purification protocol is:
Loading/Washing Buffer:  20 mM Tris-HCl, pH 7.4
                                        150 mM NaCl, 10 mM EDTA,
Elution Buffer:                  20 mM Tris-HCl, pH 7.4
                                        150 mM NaCl, 50 mM CaCl₂,

1. Pack the gel in a suitable column, approx.
    16 mm in diameter
2. Equilibrate the column with Loading Buffer at a    
    flow rate of 2 mL/min.
3. Load the sample to be purified using a flow rate
    of 0.5 mL/min.
4. Wash the column with Washing Buffer using a
    flow rate of 2 mL/min.
5. Once the A280 of the effluent has decreased to
    the baseline, elute the bound protein with
    Elution Buffer using a flow rate of 2 mL/min.
6. Dialyze the elution fraction against a suitable
    buffer (e.g. TBS, Tris Buffered Saline, pH 7.4)
    and store at –20°C.

Note: As Ca²⁺ ions in the sample may inhibit binding, the presence of a chelator such as EDTA (2-10 mM) is recommended during the binding step. In some cases, Ca²⁺ concentrations >50 mM may enhance the elution step.
 
Example
Factor X (5 µg) was purified using ADG3570Mx sepharose (500 µl, 1:1 suspension) in a spin column. The fractions were analysed by Western blot analysis using (A) the monoclonal anti-Gla antibody M3B or (B) a polyclonal anti-Factor X antibody. (1= flow through, 2-4 Wash, 5-8 Eluate, M= Protein marker)

    

Presentation
Approximately 1 mL of settled gel immobilized with 5 mg of purified antibody with approximately 1.0 mL of PBS, pH 7.4 with 0.02% NaN₃ added as a preservative.

Storage
Store the immobilized gel at 2° - 8°C. The gel should be washed thoroughly with PBS, pH 7.4 pH prior to use to remove traces of the sodium azide preservative.

Category: Research use only

Type: Antibody

Product Availability: Worldwide

Manufacturer: ImmBioMed GmbH & Co KG, Germany

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