FITC

Fluorescein isothiocyanate (FITC) is the most common fluorescent label used to modify proteins and other biomolecules (Chapter 8, section 1.1). The isothiocyanate group reacts with amines in protein molecules to form a stable thiourea linkage (Fig. 365). Avidin or streptavidin may be tagged with this reagent to yield highly fluorescent 

Thiourea Bond Formation (Green Fluorescent Probe) 

Hgiire 365 The reaction of FITC with avidin produces a fluorescent prohe via isothiourea bonds. 

Dissolve FITC (Pierce) in DMF at a concentration of 2 mg/ml. Protect from light. 

Add 50—100 p,l of the FITC solution to each milliliter of the avidin or streptavidin solution. 

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Fission product FITC [3326-32-7] Fittings Fixation Fixation wire Fixatives... 

Fig. 7. Fluorescence polarization (FP). (a) The formation of the large FITC—protein A—IgG complex which leads to a net increase in plane polarized light transmitted from the solution. Molecular weights of the protein A-FITC, IgG, and complex are ca 43,000, 150,000, and 343,000, respectively, (b) Detection of IgG by fluorescence polarization immunoassay using A, a laboratory fluorimeter where (O) represents AP = change in polarization, and B, a portable detection unit where (D) is —fiV = change in voltage (27). The field detector proved to be more sensitive than the fluorimeter.

Figure 2. Irradiation of uranyl glass microspheres (A) and FITC-labeled microbeads (B) demonstrate photobleaching and stability of both materials.

FI=fluorescein FITC=fluoresceinisothiocyanat Adoa=8-amino-3,6-cioxooctanoic acid... 

FIG. 9 Confocal laser scanning micrograph of a hollow polymer capsule. The polymer capsule was obtained from polymer multilayer-templated FDA microcrystals after removal of the colloidal core. The FDA microcrystals were coated with SDS and 11 polyelectrolyte layers [(PAH/PSS)3/PAH/ (PSS/PAH-FITC)2]. (PAH-FITC = PAH labeled with fluorescein isothiocyanate.) The microcrystal core was removed by exposure of the coated microcrystals to ethanol, causing solubilization of FDA. 

Labelling Na,K-ATPase with ATP analogues provides evidence for contribution from charged residues that are widely separated in the sequence of a subunit of Na,K-ATPase. The first indication came from ATP sensitive covalent insertion of fluorescein-isothiocyanate (FITC) into Lys ° in the a subunit [90], The strong fluorescence signal provides a convenient probe for monitoring conformational transitions in the proteins. Site-directed mutagenesis of Lys reduces the activity of... 

Lys [47,48] and the fluorescein-5-isothiocyanate (FITC) reactive site Lys [49,50], as well as a region between residue 157 and 300, which has been proposed as part of an energy transduction region [40,44]. 

The kinetically deduced existence of two classes of substrate sites may also account for the molar ratio between ATP analogs and inhibitors on the one hand and phosphoenzyme on the other hand. This ratio has been reported to be 2 1 for the ATP analogs adenylyl imido diphosphate (AMP-PNP) [135] and 2, 3 -0-(2,4,6-trinitrophenylcyclohexadienylidine)-ATP (TNP-ATP) [97], and also 2 1 for the ATP-site directed fluorescent inhibitors eosin [99] and FITC [49,50] and the transition-state inhibitor vanadate [126]. 

Fig. 4. Tentative allocation of probe binding sites within the three-dimensional structure of Ca -ATPase derived from vanadate-induced E2-type crystals. The top picture is the projection view of the Ca -ATPase down the x-axis, revealing the pear-shaped contours of ATPase molecules. The maximum length of the cytoplasmic domain to the tip of the lobe is =r65A. In the middle and bottom pictures the same structure is viewed down the x-axis, revealing the gap between the bridge and the bilayer surface and the connections between ATPase molecules in neighboring dimer chains. The proposed binding sites for lAEDANS and FITC are indicated. The bottom right picture is the same structure viewed down the y-axis. Adapted from Taylor et al. [90].

Chemical modification studies with fluorescein-5 -isothiocyanate support the proximity of Lys515 to the ATP binding site [98,113-117,212,339]. Fluorescein-5 -isothiocyanate stoichiometrically reacts with the Ca -ATPase in intact or solubilized sarcoplasmic reticulum at a mildly alkaline pH, causing inhibition of ATPase activity, ATP-dependent Ca transport, and the phosphorylation of the Ca " -ATPase by ATP the Ca uptake energized by acetylphosphate, carbamylphos-phate or j -nitrophenyl phosphate is only partially inhibited [113,114,212,339]. The reaction of -ATPase with FITC is competitively inhibited by ATP, AMPPNP, TNP-ATP, and less effectively by ADP or ITP the concentrations of the various nucleotides required for protection are consistent with their affinities for the ATP binding site of the Ca -ATPase [114,212,340]. 

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