antibody marker

Principle

Antibody biotin labeling refers to the covalent coupling of a marker (biotin) to an antibody. The labeled antibody protein reacts specifically with the substance to be detected (e.g., immune proteins) to form a multivariate complex, and automated determination of the test results is carried out with the aid of precision instruments, such as enzyme-labeled detector, flow cytometer, and other precision instruments for the qualitative and quantitative determination of the substance to be detected.

Operation method

Biotinylated labeling of antibodies

Principle

Antibody biotin labeling refers to the covalent coupling of a marker (biotin) to an antibody. The labeled antibody protein reacts specifically with the substance to be detected (e.g., immune proteins) to form a multivariate complex, and automated determination of the test results is carried out with the aid of precision instruments, such as enzyme-labeled detector, flow cytometer, and other precision instruments for the qualitative and quantitative determination of the substance to be detected.

Materials and Instruments

Antibody to be labeled, biotin (Thermo), pure water
EP tube, pipette gun, 1X PBS solution PH 7.2~7.4
Dialysis bag, BCA kit, 96-well plate, 37 ℃ constant temperature box
Shaker

Move

1. Biotin-labeled antibody

(1) According to Thermo's instructions, bring the reagents and the antibody to be labeled to room temperature, weigh a certain amount of biotin, dissolve and dilute the biotin in sterile water to a final concentration of 5.5 mg/mL.

(Example: For every 1.0 mg of biotin, 180 μL of sterile purified water is used to dissolve 5.5 mg/mL of solvent.)

(2) The mass ratio of biotin to antibody is 5-20:1, mix the calculated mass of biotin and antibody to be tested in a total volume of more than 100 μL.

(3) Rotate the reaction for 1 h at room temperature away from light.

(4) After the reaction, dialyze with 1×PBS solution at 4 ℃ overnight. On the next day, the dialysis was continued for 5 h with a new 1×PBS solution.

(5) At the end of dialysis, collect and pack, store at -80 ℃ under low light, and add 60% glycerol for long-term storage.

2. Detection of antibody concentration after labeling

(1) Determine the concentration of labeled antibody by BCA method. Dilute the standard protein (0.5 mg/mL BSA) with the standard diluent and add it into the 96-well plate according to the required gradient, the total volume is 20 μL.

(2) Dilute the antibody to be tested 10 times and add 20 μl of the dilution solution to the 96-well plate (make duplicate wells).

(3) Shake and mix the Solution A in the BCA kit, and according to the number of samples, prepare the appropriate amount of BCA working solution by adding 50 volumes of Solution A to 1 volume of Solution B (50:1), and mix thoroughly.

(4) Add 200 μL of BCA working solution into each well, blow gently with a sampling gun to mix well, and leave it at 37 ℃ for 30 min.

(5) After cooling to room temperature, determine A562 by enzyme marker.

(6) Plot the standard curve according to the result and calculate the protein concentration in the sample.

Caveat

1. The concentration of protein to be labeled should not be too low, if the concentration is too low, the concentration should be increased to more than 0.1 mg/mL by ultrafiltration and concentration.

2. if the coupling group is amino acid, the system of the marker must not contain free amino acid (Tris, amino acid), carrier protein (BSA) or other interfering substances, if there are interfering substances, it is necessary to make sure that the interfering substances are removed cleanly by ultrafiltration with labeling buffer repeatedly, and if other groups are used to couple the reagent, the system of the marker must not contain substances containing the corresponding free groups.

3. if the substance to be labeled is of small molecular weight, pay attention to the molecular weight cut-off of the ultrafiltration tube, desalting column and dialysis bag during the purification process.

4. The volume after the labeling reaction should be at least 100 μL in order to achieve the minimum sample size for purification.

5. After the labeling reaction is completed (amino-coupled system), an appropriate amount of free amino-containing small molecules can be added to the system to neutralize the free biotin-activating reagent, improve the purification efficiency, and reduce the false positives and high background of subsequent reactions.

6. The purified labeled protein can be used to determine the protein concentration by A280 or BCA method.

7. The labeled and purified proteins can be divided and stored for a long time according to the nature of the proteins, reducing the number of freezing and thawing.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/