Fluorescein-labeled antibody technology A) Principle Fluorescein currently used for antibody labeling mainly includes Fluorescein isothiocynate (FITC) or Rhodamine (Lissamine rhodamine B200, RB200). Under alkaline conditions, the carbon amide bond of FITC can be covalently bound to the epsilon amino group of antibody lysine, and the labeled antibody still retains the ability to bind to the corresponding antigen. The yellow-green fluorescence is generated under the excitation of the fluorescent lamp source ultraviolet or blue-violet light, and the corresponding antigen can be qualitatively, localized or quantitatively detected by observation under a fluorescent microscope or analysis by flow cytometry. (2) Operation steps The purified IgG antibody was dialyzed against PH9 ～ 9.5 carbonate buffer overnight. ↓ Weigh an appropriate amount of IFTC and add dimethyl sulfoxide (DMSO) (FITC ～ 1mg / 1ml DMSO) ↓ Add FITC-DMSO solution dropwise to the antibody solution after dialysis according to the above ratio ↓ The label was added to 2.5ml with PBS, and the magnetic stirrer was stirred at room temperature in the dark for 2h ↓ Remove free fluorescein with PD10 column (Sephadex G25 column), first rinse G25 column with 25ml PBS ↓ Collect the first peak of fluorescein-binding protein eluted by PBS and measure F / P Calculation: 2.87 × A495 The appropriate F / P value is 2 ~ 4. (3) Reagent equipment 1. Purified polyclonal antibody or monoclonal antibody. 2. FITC (Fluorescein-5-Lsothiocyanalte) or other fluorescent pigments. 3. PBS, DMSO 4. PH9 ～ 9.5 carbonate buffer: Na2CO34.3g, NaHCO3 8.6g, add distilled water to 500ml. 5. PD10 column (Sephadex G25 column) 6. Magnetic stirrer, ultraviolet spectrophotometer, etc. (Four) matters needing attention 1. Store FITC in a dark place at 4 ° C. Open and weigh the reagent bottle when it reaches room temperature before use to avoid deliquescence. 2. FITC-DMSO solution should be prepared immediately before use. 3. The carbonate buffer should be prepared fresh. Low Temperature Bamboo Charcoal Low Temperature Bamboo Charcoal,Black Bamboo Charcoa,Black Low Temperature Bamboo Charcoal,Bamboo Charcoa Sichuan Shihai Import And Export Trade Co.Ltd , https://www.zgshzm.com
After dialysis, the antibody solution was transferred to a small beaker
Make the final concentration 1mgFITC / 1mlDMSO
FITC / IgG ratio: if the IgG concentration is 1 mg / ml, the FITC / IgG ratio is about 50 μg FITC / mgIgG;
If IgG is 5 ～ 10mg / ml, the ratio is 25μg FITC / ml IgG
Put the antibody in the 10ml small beaker first
ratio. Free fluorescein
F ï¼ P ï¼ â”€â”€â”€â”€â”€â”€â”€â”€
A280-0.35 × A495