PRINCIPLE

Fluorescein isothiocyanate (FITC) is a widely used due to its high quantum efficiency and stability when conjugated. FITC is yellow-orange in color with an absorption maximum at 495 nm. Upon excitation it emits a yellow-green color with an emission maximum at 525 nm. Conjugation occurs through primarily-amine groups of lysine residues, forming a stable thiourea bond. Conjugation of fluorescein isothiocyanate (FITC) to antibody is an extremely valuable technique for identifying surface molecules using either fluorescence microscopy or flow cytometry. The following is the procedure that antibody molecules are coupled with fluorescein derivatives.

MATERIALS

METHODS

1. Change the antibody solution with 0.2 M Sodium carbonate-bicarbonate buffer (pH 9.0) with G-25 column.

2. Determine antibody concentration based upon A280.

3. Prepare the required dilution of FITC in 0.2 M Carbonate-bicarbonate buffer (FITC labeling buffer).

4. Add FITC labeling buffer to antibody solution and standing for overnight at 4°C.

5. Cover the reaction vial with aluminum foil to protect from light.

6. Separate conjugate from free FITC on G-25 column, and collect fractions.

7. Determine F/P ratio of conjugate spectrophotometrically.

F/P = Moles of FITC / Moles of protein

An F/P of 5 to 6:1 is usually optimal for flow cytometry.

8. Change antibody solution with Phosphate buffered saline (PBS) buffer with G-25 column.

9. Collect the labeled solution. Store at 4°C and protect from light.

HOT PRODUCTS

PDCD1 HIV1 gp120 CD33 IAV HA PRNP TNF MS4A1 CD274 APP EGFR CD40 ERBB3 TFPI ERBB2 SARS-CoV S PCSK9 MET BSG HAVCR2 IL6 CD3 LAG3 ROR1 TNFRSF4 IL33 CD19 CD38 JAG1 IL17A IFNG CD47 HCV E2 Protein IL1A / IL1B HBsAg IGF1R CD4 AXL ANGPT2 IL23A NGF MUC1 TNFRSF18 TNFRSF9 MSTN IL1B HRSV F Protein KDR SORT1 C5 VEGF CXCR4 TNFRSF17 Tau TFRC CTLA4 IL13 IAV H5N1 HA TSLP MSLN SOST
For research use only, not directly for clinical use.
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