(2) Operation steps
The purified IgG antibody was dialyzed against PH9 ~ 9.5 carbonate buffer overnight.
After dialysis, the antibody solution was transferred to a small beaker
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Weigh an appropriate amount of IFTC and add dimethyl sulfoxide (DMSO) (FITC ~ 1mg / 1ml DMSO)
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
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Add FITC-DMSO solution dropwise to the antibody solution after dialysis according to the above ratio
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The label was added to 2.5ml with PBS, and the magnetic stirrer was stirred at room temperature in the dark for 2h
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Remove free fluorescein with PD10 column (Sephadex G25 column), first rinse G25 column with 25ml PBS
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Collect the first peak of fluorescein-binding protein eluted by PBS and measure F / P
ratio. Free fluorescein
Calculation:
2.87 × A495
F ï¼ P ï¼ â”€â”€â”€â”€â”€â”€â”€â”€
A280-0.35 × 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.
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