How do you label peptides?
Three strategies are used to label peptides with dyes:
- Labeling during synthesis of peptide.
- Synthetic peptides can be covalently modified on specific residues and labels incorporated following synthesis.
- Synthetic peptides may be covalently labeled by amine- or thiolreactive protein labels.
How do I create a FITC label?
Dissolve FITC in anhydrous DMSO at 1 mg/ml concentration. This should be prepared fresh for each labelling reaction. Add 5 μl of the dye to the dialysed protein (5 ml) every 30 min to get a final concentration of 100 ng FITC/1 μg protein. Perform this step by gently rocking the protein solution at 4°C for 12 h.
How does FITC labeling work?
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 do you use FITC?
Covalent conjugation. 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.
What Colour is FITC?
FITC exhibits an excitation maximum at λ = 495 nm and emission maximum at approximately λ = 519 nm. The color of the compound is yellow while the emitted light is green.
How do you measure FITC?
The easiest way to determine the FITC to Antibody (F/A) ration, meaning how many FITC molecules you have on average on one molecule, or antibody concentration you can use a spectrometer (NanoDrop for example) and measure the absorbance at the wavelengths 280 and 495 nm.
Why is FITC used?
Fluorescein-5-isothiocyanate (FITC) is a fluorescence dye and belongs to the xanthene dyes. FITC is used for labeling of different biomolecules, e.g. immunoglobulins, lectins and other proteins, peptides, nucleic acids, nucleotides; oligo-and polysaccha- rides.
What is FITC staining used for?
How do you read a peptide sequence?
The primary structure (or sequence) of a peptide or protein is always written starting with the amino terminus on the left and progressing towards the carboxy terminus.
Which peptides can be conjugated with FITC?
Scheme of FITC labeling peptides on solid support (N-terminal β-alanine). N-terminal amino group can be selectively conjugated with FITC in the presence of the side chain amino groups due to their different pKa values (approx. 8.9 and 10.5, respectively).
What are the requirements for peptide cleavage in FITC?
Perform standard peptide cleavage. FITC does not require any specific additive. FITC can undergo photobleaching and therefore it is recommended to protect FITC reagent and FITC-peptide/protein conjugates from light. Figure 2. Scheme of FITC labeling peptides on solid support (N-terminal β-alanine).
What is FITC used for in biochemistry?
Fluorescein isothiocyanate (FITC) is an amine reactive derivative of fluorescein dye that has wide ranging application in biochemistry. It has been extensively used to label peptides and proteins. However, its use in solid phase peptide synthesis is restricted.
What is n-ter FITC used for in solid phase peptide synthesis?
It has been extensively used to label peptides and proteins. However, its use in solid phase peptide synthesis is restricted. Indeed, in acidic conditions required for linker cleavage, N-ter FITC-labeled peptides undergo a cyclization leading to the formation of a fluorescein with subsequent removal of the last amino acid.