Rapid Labeling Kit (HRP)

Cat. No.: R1521479

Übersicht

Intended Use

For the conjugation of antigens and antibodies with horseradish peroxidase (HRP), featuring simplicity and rapidity.

Reaction Principle

The lyophilized horseradish peroxidase in the kit is activated by sodium periodate, resulting in a large number of free aldehyde groups. These aldehyde groups can directly react with amino groups on antigens and antibodies under weakly alkaline conditions (adjusted by the initiator) to form Schiff bases, which can be reduced by a reducing agent to form stable chemical bonds.

Storage Conditions and Shelf Life

1、The kit shall be protected from strong light. It is valid for 6 months when stored at 2–8℃, and 18 months when stored at -20℃.

2、Once opened, the lyophilized HRP vial can be used for 2 days if stored at 2–8℃.

3、Other components of the kit shall be promptly returned to 2–8℃ after use, and must be used within the kit’s shelf life.

Requirements for Analytes to Be Labeled

1、If sodium azide is used as a preservative, the material must be dialyzed against 0.01 M PBS (pH 7.4).

2、Protein materials containing amino groups such as urea, glycine, Tris, and ammonium sulfate in the solvent must be dialyzed against 0.01 M PBS (pH 7.4) prior to labeling.

3、Low purity of the analyte will compromise labeling efficiency; further purification of the analyte is recommended.

4、Precipitates in the analyte may affect labeling efficiency. It is recommended to remove them by high-speed centrifugation or filtration, and use only after confirming no deterioration.

Labeling Protocol

A. Experimental Preparation

a) Remove the kit from refrigerated or frozen storage, equilibrate at room temperature (18–25℃) for 20–30 minutes. Allow liquid components to fully thaw and reach room temperature, then vortex to mix before use.b) The recommended antibody concentration is 0.5 mg/mL–5.0 mg/mL.c) For antigen labeling, optimization of the optimal labeling ratio is required.

B. Experimental Procedure

a) Antibody Preparation: Before use, dilute (or concentrate) the antibody with 0.01 M PBS (pH 7.4) to the specified concentration listed in the appendix.

b) Conjugation: Add the antibody to the lyophilized enzyme, followed by the corresponding volume of initiator. Vortex thoroughly until the lyophilized enzyme is completely dissolved, then incubate at room temperature in the dark for 2 hours.

c) Reductant Preparation: Add 60 μL of initiator to 1400 μL of purified water and vortex to mix. Pipette 1 mL of this diluted solution into the reductant to obtain a 10 mg/mL reductant solution.

d) Reduction: Immediately add the corresponding volume of reductant solution to the reacted enzyme-labeled conjugate, and incubate at room temperature in the dark for 2 hours.

e) Termination: Add the corresponding volume of stop solution, mix well, and incubate at room temperature in the dark for 10 minutes.f) Storage: Add an equal volume of glycerol, mix thoroughly, and store at -20℃.

Operating Procedure (Example: Labeling 0.2 mg of antibody at a concentration of 5 mg/mL)

Precautions

1、Carefully read the instruction manual before operation, and perform the test strictly in accordance with the kit instructions.

2、Avoid conducting tests in harsh environments (such as environments containing corrosive gases and dust like 84 disinfectant, sodium hypochlorite, acid-base, or acetaldehyde).

3、The reducing agent must be prepared freshly for immediate use; dissolve it with the initiator dilution and use promptly. Discard the reducing agent immediately with the cap open after use.

4、Wear disposable gloves when handling reagents, and wash hands thoroughly after operation.

5、Please use the reagents within their validity period.

Appendix (Taking 2 mg/vial lyophilized HRP as an example):

antibody concentration（mg/ml）	Amount of antibody（ml）	Amount of initiator （ml）	Amount of reducing solution（ml）	Amount of stop solution （ml）	Amount of glycerol（ml）
0.5	4.0	0.200	0.200	0.600	5.00
1.0	2.0	0.115	0.150	0.450	2.715
2.0	1.0	0.075	0.100	0.300	1.475
3.0	0.7	0.062	0.100	0.300	1.162
4.0	0.5	0.055	0.100	0.300	0.955
5.0	0.4	0.050	0.100	0.300	0.850

Note: Based on the amount of lyophilized HRP, the dosage of each component can be increased or decreased proportionally.

Lagerung und Versand

Storage

Protected from light,Store at -20°C

Verschickt in

Ice chest + Ice pads

Contents & Storage

A1519981	Name	small package	big package
A1519981A	Lyophilized horseradish peroxidase (Lyophilized HRP)	0.2mg×5	1mg×5
A1519981B	Initiator	1ml×1	1ml×1
A1519981C	Reducing Agent	10mg×5	10mg×5
A1519981D	Stop Solution	0.5ml ×1	1ml ×1