Proteins FITC labeling

In addition Choi et al. utilized FlTC-labeled methamphetamine for competitive immunoassay of methamphetamine in urine (19). Instead of purified antibody or antibody fragment, antiserum was used. An aminobutyl derivative of metamphetamine was conjugated with proteins and used as an immunogen to produce antibodies for the assay. The free FITC-labeled tracer was well separated from the antibody-bound fraction, and the detection limit for the CE assay was lower than that for ELISA. 

The solution diffusion properties of FITC-labelled BSA were measured by FRAP [12], The results showed that the protein diffused freely in solution with a diffusion coefficient of approximately 3xl0 7 cm2/s. This was in reasonable agreement with previously published values [36]. FRAP measurements were also made on thin films stabilized by FITC-BSA. The films were allowed to drain to equilibrium thickness before measurements were initiated. Thin films covering a range of different thicknesses were studied by careful adjustment of solution conditions. BSA stabilized films that had thicknesses up to 40 nm showed no evidence of surface diffusion as there was no return of fluorescence after the bleach pulse in the recovery part of the FRAP curve (Figure 14(c)). In contrast, experiments performed with thin films that were > 80 nm thick showed partial recovery (55%) of the prebleach level of fluorescence (Figure 14(b)). This suggested the presence of two classes of protein in the film one fraction in an environment where it was unable to diffuse laterally, as seen with the films of thicknesses < 45 nm, and a second fraction that was able to diffuse with a calculated diffusion coefficient of lxlO 7 cm2/s. This latter diffusion coefficient was 3 times slower than that... 

Figure 8.5 Pomegranate by-product (PBP) consumption by E° mice attenuates atherosclerotic lesion development, in association with reduction in macrophage oxidative stress and Ox-LDL uptake. E° mice consumed PBP (17 or 51.5 mg gallic acid equivalents/kilogram/day) for 3 months. Control mice received only water (placebo). At the end of the study, the mice aortas as well as the mice peritoneal macrophages were harvested. (A) Atherosclerotic lesion size determination. (B) Total macrophage peroxide levels were determined by the DCFH-DH assay. (C) For determination of macrophage paraoxonase 2 (PON2) lactonase activity, cells (2 x 10e) were incubated with 1 mmol/L dihydrocoumarin in Tris buffer, and the hydrolysis rate was determined after 10 min of incubation at 25°C. (D) The extent of Ox-LDL (25 pg of protein/ milliliter, labeled with FITC) uptake by the mice macrophages (1 x 10e) was determined by flow cytometry. Results are expressed as mean S.D. of three different determinations. = p < 0.01 versus placebo.

An IEF/CGE separation of proteins (GFP, FITC-labeled ovalbumin) was carried out on an acrylic chip. EK mobilization at - 20 pm/s allows repeated injections of the focused bands into the cross-channels for CE separations. Even though the ampholyte entered into the CE channel, normal CE occurred at a... 

In labeling amino acids, peptides, and proteins, FITC can only label primary and secondary, but not tertiary, amino groups. Why (2 marks)... 

There are several recent examples of the switching of nonspecific protein binding on polymer surfaces by application of an external stimulus. Alexander and coworkers demonstrated that protein adhesion can be controlled on PNIPAM surface brushes [14, 181]. For instance, it was reported that the adsorption of FITC-labeled bovine serum albumin (FITC-BSA) on PNIPAM/hexadecanethiol micropatterned surfaces could be tuned by LCST. However, this effect was found to be less pronounced after prolonged incubation times or repeated heating/cooling cycles. The authors suggested that this behavior could be due to unspecific PNIPAM-protein interactions [14],... 

Surface-exposed parts of the protein have been distinguished by their ability to bind specific antibodies and proteolytic enzymes under nondenaturing conditions (see Figure 16). Many of these epitopes are located at positions in the sequence that correspond to the external side of the suggested nucleotide-binding fold (Mate et al., 1992). Antibodies to fluorescein bind only to denatured FITC-labeled Ca2+-ATPase, and not to the native FITC-labeled enzyme. This is consistent with a location of bound FITC in a hydrophobic cleft corresponding to the ATP site. 

Figure 13 Molecular-mass analysis of phosphorylase B (panel B) and the separation of the noncovalently (Sypro Red) labeled protein markers (panel A ALA, a-lactalbumin CBA, carbonic anhydrase OVA, ovalbumin BSA, bovine serum albumin BGA, (f-galactosidase) and the covalently (FITC) labeled protein markers (panel C TRI, tripsin inhibitor CAH, carbonic anhydrase ADH, alcohol dehydrogenase BSA, bovine serum albumin BGA, P-galactosidase). Separation conditions gel, 1% agarose, 2% linear polyacrylamide (LPA, MW 700,000-1,000,000) in 50 mM Tris, 50 mM TAPS, 0.05% SDS (pH 8.4) separation buffer, 50 mM Tris, 50 mM TAPS, 0.05% SDS (pH 8.4) separation voltage, 420 V, current, 5 mA gel thickness, 190 pm effective separation length, 3.5 cm temperature, 25°C sample loading, 0.2 pL into 2.5 x 4 x 0.19-mm injection wells. Sample buffer contained 0.05% SDS and 1 x Sypro Red. (Reproduced with permission from Ref. 141.)...

In some eases one of these two counterjarts is additionally labeled with a radionuclide, chromophore, fluorophore, enzyme, etc. to facilitate the detection and to make the method more sensitive. Leucoagglutinin labeled with tritium and protein A labeled with FITC can serve as examples. 

Protein A binds to the F,. region of IgG and does not interfere with the antigen binding capacity of IgG, thus FITC-Protein A can form tertiary complexes with antigen and antibody (IgG) . FITC-labeled protein A has been applied in different immunochemical studies and separations of cells bearing IgG or other... 

The principles of fluorescence quenching have been successfully applied to assays for protease activity. Proteases are digestive enzymes that degrade polypeptides into smaller oligopeptides or constituent amino acids. A general assay12 for protease activity employs a substrate prepared by covalently derivatizing a protein, transferrin, with a number of fluorescein isothiocyanate (FITC) labels. The FITC labeled proteins (Fig. 3.3) exhibit absorbance maxima at 495 nm and emission maxima at 525 nm. 

The AcCh biosensor uses competitive binding between FITC labeled or unlabeled bungarotoxin and receptor immobilized by adsorption onto the surface of a quartz optical fiber. Once bound to the protein BTX, FITC exhibits absorption and emission maxima of 495 nm and 520 nm, respectively. In the absence of analyte (BTX), a maximal quantity of FITC-BTX binds to the immobilized receptor protein. 

Fig. 1. Binding of protein L to human B cells and to mouse B cells expressing human Igs. a Human peripheral blood lymphoid cells were stained with FITC-labeled anti-human CD 19 monoclonal antibody and biotinylated protein L, followed by Cy-chrome-labeled streptavidin. b Splenic cells from transgenic mice expressing human Igs [46] were stained with FITC-labeled anti-mouse B220 monoclonal antibody and biotinylated protein L, followed by Cy-chrome-labeled streptavidin. All monoclonal antibodies and their corresponding isotype controls were purchased from BD PharMingen.

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