PEROXIDASE LABELING KIT

General description

The peroxidase labeling kit is responsible for the labeling of primary amino groups of biomolecules with activated peroxidase (POD) from horseradish.

Specificity

Purity number: (A405nm/A275nm): 3.0 – 3.5.
Isoenzyme distribution: > 90% homogeneous isoenzyme C.
Assay Time: The procedure is developed for a reaction temperature of +25°C. At +25°C the reaction time should be 2 hours but it can be extended to 3 hours without influencing the results. Alternatively, the reaction can be carried out at +2 to +8°C with a reaction time of 18 hours up to 24 hours.

Application

The peroxidase labeling kit is designed for labeling water-soluble biomolecules containing 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 with peroxidase, as the resulting conjugate is optimal for use in immunochemical detection systems like:

• ELISA (enzyme-linked immunosorbent assay)
• immunohistochemistry
• immunoblotting

Features and Benefits

• Fast - the whole labeling procedure can be performed in one day
• Easy - only 5 simple working steps
• Complete - all necessary buffers and reagents are supplied with the kit

Packaging

1 kit containing 7 components.

• Peroxidase (activated)
• Sodium Carbonate/hydrogen Carbonate Buffer
• Sodium Borohydride (tablets)
• Triethanolamine Buffer
• Glycine Solution
• Dialysis Buffer
• Stabilizing Agent (includes BSA and Kathon CG)

Preparation Note

Working solution: Preparation of working solutions

For best results, mark each solution with the appropriate number. Volumes indicated are sufficient to label a portion of 1.2 mg IgG.

Solution 1: Peroxidase (activated)
Reconstitute the lyophilizate of bottle 1 in 0.5 ml double-distilled water (peroxidase concentration = 16 mg/ml).

Solution 2: Sodium Carbonate/-hydrogencarbo-nate Buffer (100 mM, pH 9.8)
Equilibrate bottle 2 to 15 to 25 °C. Be sure that all buffer components are dissolved. Add 10 ml of bottle 2 to 90 ml double-distilled water, mix well.

Solution 3: Sodium Borohydride Solution (200 mM)
We recommend to wear gloves when working with sodium borohydride. Prepare the solution immediately prior to use and keep cold on ice. Add 1 tablet of bottle 3 to 130 ml cold, double-distilled water, mix well.

Solution 4: Triethanolamine Buffer (2 M, pH 8.0)
Bottle 4, ready-to-use. Equilibrate the bottle to 15 to 25 °C. Be sure that all buffer components are dissolved.

Solution 5: Glycine Solution (1 M, pH 7.0)
Reconstitute the lyophilizate of bottle 5 in 0.5 ml double-dist. water (glycine concentration = 1 M)

Solution 6: Dialysis Buffer (1x conc.)
Equilibrate bottle 6 to 15 to 25 °C. Be sure that all buffer components are dissolved. Add 30 ml of bottle 6 to 570 ml double-dist. water, mix well.

Solution 7: Stabilizing Agent
Bottle 7 is ready-to-use. Equilibrate the bottle to 15 to 25 °C.

Preparation of the immunoglobulin solution
The IgG concentration of the antibody solution should be approx. 4 mg/ml (3.8 to 4.2 mg/ml). This concentration is critical for the coupling and should hence be checked photometrically before every coupling and adjusted if necessary: 1 mg/ml = 1.40 at A_{280 nm} and 1 cm path length. 0.3 ml of this solution are required for each labeling reaction.
Note: Do not use preservatives, such as sodium azide and stabilizers, such as albumin or detergents.

Lyophilized immunoglobulin, salt-free
Weigh 1.6 mg into a suitable vessel and dissolve in 0.4 ml solution 2. Check the concentration and pH and adjust, if necessary.

Immunoglobulin in buffer

• When immunoglobulin is dissolved in phosphate buffered saline (PBS) without additional proteins or preservatives: Adjust the pH to 9.8 with 1 M sodium carbonate buffer (bottle 2). If necessary, dilute with solution 2 to obtain an IgG concentration of 4 mg/ml.
• When the immunoglobulin is dissolved in a buffer with organic salts: Dialyse immunoglobulin with solution 2 and adjust the concentration to 4 mg/ml with solution 2.

• Solution 1 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; note that a loss ofactivity of 10 to 20% may be observed.
• Solutions 2, 5 and 6 are stable for one week at 2 to 8 °C, and for 6 months at -15 to -25 °C, when stored frozen in aliquots.
• Solution 3 should always be prepared immediately before use.
• Solution 4 and 7 are stable at 2 to 8 °C until the expiration date indicated on the kit.

Other Notes

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