PEROXIDASE (POD) ACTIVATED

General description

Donor: hydrogen-peroxide oxidoreductase
Reagent for the labeling of water-soluble substances carrying primary amino groups with peroxidase from horseradish (HRP).
Capacity: sufficient for approximately 6 mg IgG.

Peroxidase (POD) is an enzyme that includes a group of specific enzymes such as NAD peroxidase, NADP peroxidase, fattyacid peroxidase and a group of non specific enzymes from various sources. It is present in animals, higher plants and other organisms.

Application

Peroxidase (POD), activated has been used in terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining and immunohistochemistry.

The enzyme can be used without pre-activation for labeling water-soluble substances with reactive and accessible primary amino groups (e.g., peptides or proteins) with peroxidase for use in analytical methods. It is particularly suitable for the coupling of antibodies (Ig, Ig Fab, and Ig F(ab′)₂ fragments from rabbit, mouse, sheep, and goat) with peroxidase. The resulting conjugate is optimal for use in immunochemical detection systems, for example, ELISA, immunohistochemistry, and immunoblotting procedures.

Biochem/physiol Actions

Peroxidase (POD) helps in the dehydrogenation of several organic compounds like phenols, aromatic amines, hydroquinones etc.

Quality

Isoenzymes: >90% isoenzyme C (HPLC)
Purity number: 3.0 - 3.5 (A₄₀₃/A₂₇₅)

Preparation Note

Stabilizers: Product is stabilized with sucrose.
Working concentration: 1:4,000 to 1:10,000
For ELISA
Working solution: Preparation of the solutions, (15 to 25 °C)

1. 1 M Sodium carbonate/-hydrogencarbonate solution, pH 9.4
1 M Na₂CO₃: Dissolve 10.6 g Na₂CO₃ in 80 ml double-dist. water and make up to 100 ml.
1 M NaHCO₃: Dissolve 8.4 g NaHCO₃ in 80 ml double-dist. water and make up to 100 ml.
Adjust the pH of the NaHCO₃ solution to 9.4 by adding Na₂CO₃ solution.

2. 100 mM Sodium carbonate/-hydrogencarbonate solution, pH 9.8
Dilute 10 ml solution 1 to 100 ml with double-dist. water.

3. 200 mM Sodium borohydride solution
NB: Prepare the solution immediately prior to use and keep cold on ice. Dissolve 8 mg NaBH₄ in 1 ml cold double-dist. water.

4. 2 M Triethanolamine solution, pH 8.0
Dilute 2.66 ml triethanolamine with 3 ml double-dist. water, adjust the pH to 8.0 with 25% HCI and make up to 10 ml with double-dist. water.

5. 1 M Glycine solution, pH 7.0
Dissolve 0.75 g glycine in ca. 6 ml double-dist. water, adjust to pH 7.0 with 0.1 M NaOH, and make up to 10 ml with double-dist. water.

6. PBS (phosphate-buffered saline); glycine; pH 7.4.
10 mM Potassium phosphate, 200 mM NaCI, 10 mM glycine, pH 7.5.

• Solution A (K₂HPO₄): Dissolve 4.56 g K₂HPO₄ × 3 H₂O, 23.4 g NaCI, and 1.5 g glycine in ca. 1,500 ml double-dist. water and make up to 2000 ml with double-dist. water.
• Solution B (KH₂PO₄): Dissolve 2.72 g KH₂PO₄, 23.4 g NaCI, and 1.5 g glycine in ca. 1,500 ml double-dist. water and make up to 2,000 ml with double-dist. water.
• PBS: Whilst controlling pH, add sufficient solution B to solution A until the pH is 7.4.

7. Antibody solution

0.3 ml required for each labeling reaction. The IgG concentration of the solution to be used is c = 4 mg/ml (3.8–42 mg/ml). This value is critical for the coupling and hence should be checked photo-metrically for every test and adjusted if necessary: A_280nm, 1cm, 1 mg/ml = 1.40.
NB: Do not use preservatives (e.g., sodium azide) and stabilizers (e.g., albumin).

• Immunoglobulin, salt-free, lyophilized:

Weigh 1.6 mg into a suitable vessel and dissolve in 0.4 ml solution. Check the concentration and pH and correct if necessary.

• Immunoglobulin in buffer:

PBS buffer without additional proteins or preservatives: adjust the pH to 9.8 with solution 1 and if necessary dilute with solution 2 to obtain an IgG concentration of 4 mg/ml. Buffer with organic salts: Dialyze immunoglobulin into solution 2 and adjust the concentration to 4 mg/ml with solution 2.

Stability of the solutions
Solutions 1, 2, 4, 5 and 6 are stable for 1 week at 2 to 8 °C. Solutions 3 and 7 should always be prepared immediately prior to use.

Storage conditions (working solution): The reconstituted solution is stable for 3 months at 2 to 8 °C. The solution can be aliquoted and shock-frozen at -60 °C or below, and then stored at -15 to -25 °C; a loss of activity of 10–20 % can result.

Reconstitution

Dissolving the lyophilizate in 0.5 ml double-dist. Water produces a peroxidase concentration of 16 mg/ml.

Other Notes

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