Acridine esters (NSP-SA-NHS), Cas199293-83-9 and related compounds have been proven to be very advantageous chemiluminescent markers, whose stability, activity and sensitivity exceed certain radioisotopes. Acridine esters can react with proteins containing primary amino groups. Under alkaline conditions, NHS is substituted as a leaving group, and the protein forms a stable amide bond with the acridine ester. After the reaction is completed, the excess acridine salt is removed by the desalting column. The instructions for use of NSP-SA-NHS are described below (for example, antibody/protein labeling).

1. Preparation

Labeling buffer: 0.2M NaHCO3 (pH=9.0);

Marking stop buffer: 10% Lysine 0.2M NaHCO3 (pH=9.0) solution;

Desalting purification buffer: 0.1M Na2HPO4-NaH2PO4 (pH=6.5);

Acridine ester luminescent liquid: (0.01~0.1M) NaOH+0.05% H2O2.

2. Calculate the amount of antibody (protein) and acridinium used for labeling:

Usually set the molar ratio n (antibody): n (acridine ester) = 1:10~1:50, and the specific needs to be adjusted according to the amino acid composition;

Prepare 2.5mg/ml acridinium ester-DMSO mother liquor, keep it in glassware and avoid light;

Use 0.2M NaHCO3 (pH=9.0) solution to prepare 0.1~0.5 mg/ml antibody reaction solution.

3. Connection and purification

This specification exemplifies that the molecular weight of the labeled antibody antibody is 180kd corresponding to 15 times the acridinium ester. If the user's sample to be labeled has other molecular weights, please refer to Appendix 1 to change the reagent ratio.

1. Take 10 µL of diluted acridinium ester mother solution (2.5mg/ml), add 90 µL of anhydrous DMSO (or DMF) and dilute it 10 times to prepare acridinium ester working solution (0.25mg/ml).

2. Use 0.2M NaHCO3 (pH=9.0) solution to dilute 50 µg of antibody to 300 µL, and add 10 µL of acridinium ester working solution (0.25 mg/ml) (refer to Appendix 1).

3. Wrap it in tin foil and mark it at room temperature for 0.5~1h.

4. Quench the reaction, add 100 µL of labeling stop buffer, and mix at room temperature for 30 minutes (Note: If the labeling is sufficient, you can skip this step and proceed directly to column purification).

5. Purification by desalting column, collecting acridine-labeled protein components and detecting the concentration, please refer to the back of the manual.

6. To detect the labeled protein, take 10 µL of acridine-labeled protein and place it in a black 96-well plate. Inject 50 µL~150 µL of acridine ester luminescent solution into each microwell, and detect immediately. If the luminescence is too strong, the labeled protein can be detected. Dilute several times to make it within the luminescence detection limit of the instrument. Note: The acridine ester luminescent solution is best to be prepared immediately, or the NaOH solution and hydrogen peroxide solution are respectively prepared into 2x mother liquor, and then mixed 1:1 when using.

7. The acridinium ester-labeled protein can be stored in acidic buffer at 4°C and protected from light (note the addition of preservatives such as sodium azide). If the storage effect is not good, please store at -20°C or -80°C in the dark .

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