Fluorescently labeled proteins detection

Detect the fluorescent-labeled protein using a laser-based fluorescent scanner. An experimental example is shown in Fig. 3 see Note 23). 

The FRAP apparatus can also be used in a semi-quantitative manner to measure the surface concentration and subsequent competitive displacement of adsorbed labelled species, such as the fluorescent-labelled protein in the adsorbed layer of a/w or o/w thin films [10]. This can be achieved by focusing the low power 488 nm beam on the film and detection of the emitted fluorescence using the FRAP photon counting photomultiplier. The detected fluorescence signal is proportional to the amount of adsorbed protein at the interfaces of the thin film provided that the incident laser intensity is kept constant. Calculations have proved that the contributions from non-adsorbed protein molecules in the interlamellar region of the film are negligible [12],... 

The imaging hardware needs to be sensitive enough to detect minimally labeled proteins. Because a cooled, coupled charge device (CCD) had been used previously to image fluorescently labeled proteins in the gel, we decided to use a scientific grade CCD camera. CCD cameras without cooling do not have the requisite sensitivity of low noise capabilities. 

Foote and coworkers [120] developed a microfabricated system with the ability to electrophoretically preconcentrate fluorescently labeled proteins prior to their separation (see Fig. 6). The authors were able to preconcentrate the proteins using a porous silica membrane situated between adjacent microchannels that allowed for the passage of buffer ions, but excluded larger migrating molecules, such as proteins. Preconcentration factors of 600-fold were achieved using this on-chip format followed by an electrophoretic separation of proteins with SDS-PAGE. Using this chip, fluorescently labeled ovalbumin was detected at concentrations as low as 100 fmol by a combination of field-amplified injection and preconcentration at the membrane prior to microchip electrophoresis. 

FRAP (fluorescence recovery afterphotobleaching) is a complementary technique to FLAP. In FRAP, a specific cellular region is bleached after irradiation of a beam of laserand protein diffusion of a fluorescently labeled protein can be determined by detecting fluorescence recovery in the bleached area. 

The brush-gel polymer film is a submicrometer particulate layer with cross-linked polymer brushes. Nonporous particles with diameter of 350 nm, 700 nm, or 900 nm, modified with polyacrylamide, were applied on a silicon wafer as slurry to obtain a 15-pm thin layer. Coating with polyacrylamide brushes increased the capillary force for increased mobile-phase velocity and the overall separation performance. Three fluorescence-labeled proteins were separated with a sinapinic acid containing mobile phase for subsequent detection via MALDl [35], A second modification of this brush-gel polymer was manufactured by cross-linking poly(glycidyl methacrylate) and di(ethylene glycole)dimethacrylate to graft the thin layer covalently on the glass substrate and to manipulate the separation characteristic. The separation of a fluorescent dye mixture was performed on this layer and the ability for a multiple reuse was shown [36]. 

Extremely high dynamic range and exquisite sensitivity is produced by laser-induced fluorescence of FQ-labeled proteins. The dynamic range exceeds 250,000, and the detection limit is in the high yoctomole range for FQ-labeled proteins. 

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be labeled with fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissues for the presence of particular antigens, and then detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more... 

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