FITC labeled antibody - Marsshall's method

FITC labeled antibody - Marsshall's method

material:

Antibody globulin solution, 0.5 mol/L pH 9.0 carbonate buffer, sterile saline, fluorescein isothiocyanate, 1% aqueous solution of thimerosal, conical flask (25-50 ml), ice and ice trough (or 1000 ml) Beaker), electromagnetic stirrer, sterilized straw, dialysis bag, glass rod, cotton thread and beaker (500ml), 0.01mol/L PBS with pH 7.2 or 8.0, etc.

Method and steps:

1. Preparation of antibodies. Take an appropriate amount of globulin solution of known concentration, place it in a conical flask, add physiological saline and carbonate buffer to make the final globulin concentration 20mg/ml, and the buffer capacity is 1/10 of the total amount. Mix well. The triangular flask is placed in the ice trough, the electromagnetic stirring speed is appropriate, that is, the foam is preferably stirred for 5-10 min;

2. Preparation of fluorescein. According to the total amount of protein to be labeled, the desired fluorescein isothiocyanate powder is accurately weighed by using an analytical balance at a ratio of 0.01 mg of fluorescein per mg of protein;

3. Combine (or mark). The weighed fluorochrome is slowly added to the globulin solution, and the mixture is continuously stirred to avoid binding the fluorescein to the wall of the flask or the stirring glass rod, and the addition is completed within about 5-10 minutes. Then continue stirring for 12-18 h. Since it is required to keep the protein solution at about 4 ° C during the binding of fluorescein to globulin, it is necessary to add ice or remove water. The binding device can also be placed in a 4 ° C refrigerator or ice storage;

4. Dialysis. After the combination of fluorescein and globulin, the globulin solution was centrifuged at 2500 r/min for 20 min to remove a small amount of the precipitate, which was then placed in a dialysis bag and placed in a beaker at pH 8.0 at 0-4 °C. Dialysis of the buffered saline solution overnight;

5. Pass the column. The dialyzed overnight label was passed through a glucose gel G-25 or G-50 column to separate the free fluorescein, and the labeled fluorescent antibody was collected for identification;