What is FITC labeling?

Fluorescein isothiocyanate (FITC) is widely used to attach a fluorescent label to proteins vi a the amine group. The isothiocyanate group reacts with amino terminal and primary amines in proteins. It has been used for the labeling of proteins including antibodies and lectins.

How is degree of labeling measured?

Calculate Degree of Labeling Remove all excess, unbound dye by dialysis or gel filtration. Measure absorbance at 280nm and the wavelength max (Amax) in a cuvette with a 1cm pathlength. NOTE: If absorbance is >2 then dilute the solution to achieve values <2.0.

How do you use FITC?

FITC is covalently coupled to primary amines (lysines) of the immunoglobulin. Dissolve 10 mgs (the entire contents of 1 vial; no need to weigh) of FITC in 1 mL anhydrous DMSO immediately before use. Add FITC to give a ratio of 40-80 µg per mg of antibody; mix immediately.

Why is FITC used?

Fluorescein isothiocyanate (FITC)-labeled antibodies are widely used as primary antibodies in the tissue cross-reactivity (TCR) studies for the development of therapeutic antibodies.

What is PE in flow cytometry?

R-PE is a large molecule used for fluorescence-based detection, primarily in flow cytometry, microarray assays, ELISAs, and other applications that require high sensitivity but not photostability.

What Colour is FITC?

green
FITC has excitation and emission spectrum peak wavelengths of approximately 495 nm and 519 nm, giving it a green color.

What is degree of labeling?

The Degree of Labeling (DOL), sometimes called Degree of Substitution (DOS), is a particularly useful parameter for characterizing and optimizing bioconjugates, such as fluorophore-labeled proteins. It is expressed as a molar ratio in the form of label/protein.

What is f p ratio?

In flow cytometry, the effective F/P ratio is the average apparent number of fluorochrome molecules conjugated per primary antibody, as determined through fluorescence measurements taken on the flow cytometer.

What does DAPI stain for?

A simple-to-use fluorescent stain, 4′,6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.

What wavelength is DAPI?

Fluorescence properties When bound to double-stranded DNA, DAPI has an absorption maximum at a wavelength of 358 nm (ultraviolet) and its emission maximum is at 461 nm (blue). Therefore, for fluorescence microscopy, DAPI is excited with ultraviolet light and is detected through a blue/cyan filter.

What is FITC PE?

The FITC / PE Compensation Standard is to be used in conjunction with hardware or software to remove spectral overlap from fluorochromes into secondary fluorescence detectors of a flow cytometer. The FITC/PE Compensation Standard is a mixture of 4 populations of microspheres: FITC, PE, FITC/PE, and AutoFluor™.

How is protein labeling done with FITC-ispybio?

Protein labeling with FITC. About the protocol. This protocol describes how proteins can be label with FITC, suitable for runs in LigandTracer Green. Note that FITC is typically conjugated via primary amines (i.e. lysines) and can affect the binding abilities of the protein. It is critical that sodium azide is completely removed from any antibody.

Can a protein be label with FITC in ligandtracer Green?

Protein labeling with FITC About the protocol This protocol describes how proteins can be label with FITC, suitable for runs in LigandTracer Green. Note that FITC is typically conjugated via primary amines (i.e. lysines) and can affect the binding abilities of the protein. It is critical that sodium azide is completely removed from any antibody.

How does the FITC antibody labeling kit work?

Protein Labeling & Crosslinking Description Pierce FITC Antibody Labeling Kit Excitation/Emission 494⁄518 Labeling Scale 0.5-1 mg Labeling Target Antibodies (General), Proteins (General) Label or Dye Fluorescein (FITC)

What’s the best FITC ratio for protein labeling?

Final molar F/P ratio was 0.06. In another protocol it is recommended to add 15-20 ug of FITC per 1 mg of protein. I took 25 ug. The F/P ratio is 0.2. It is recommendetd for IgG F/P ratio to be 2-4, but for such small proteins as mine I think it should be no more then 1.