Peroxidase (POD), activated

Description

• General description: Donor: hydrogen-peroxide oxidoreductaseReagent for the labeling of water-soluble substances carrying primary amino groups with peroxidase from horseradish (HRP).Capacity: sufficient for approximately 6 mg IgG.
• Application: Peroxidase (POD), activated has been used in terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining[2][3] and immunohistochemistry.[4]
• Biochem/physiol Actions: Peroxidase (POD) helps in the dehydrogenation of several organic compounds like phenols, aromatic amines, hydroquinones etc.[1]
• 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 1:10,000For ELISAWorking solution: Preparation of the solutions, (15 to 25 °C)1. 1 M Sodium carbonate/-hydrogencarbonate solution, pH 9.41 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 9.8Dilute 10 ml solution 1 to 100 ml with double-dist. water.3. 200 mM Sodium borohydride solutionNB: 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 8.0Dilute 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 7.0Dissolve 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 solution0.3 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: A280nm, 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 solutionsSolutions 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.