Fluorescently labeled proteins

Bastiaens, P. I. and Jovin, T. M. (1996). Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein fluorescent-labeled protein kinase C beta I. Proc. Natl. Acad. Sci. USA 93, 8407-12. 

Flora K., Brennan J., Comparison of formats for the development of fiber-optic biosensors utilizing sol-gel derived materials entrapping fluorescently-labelled protein, Analyst 1999 124 1455-1462. 

An alternative protocol for labeling sulfhydryl-containing proteins that does not require DTT reduction can be found in a method adapted from Ando (1984). When preparing any fluorescently labeled protein, optimization of the dye-to-protein ratio is important to obtain the best performance in the intended application. 

A general protocol for the use of DCIA for fluorescently labeling proteins that contain sulfhydryl residues may be obtained by following the method discussed for AMCA-HPDP (previous section). After purification of the labeled protein, the F/P ratio of fluorophore incorporation may be determined by measuring its 382nm/280nm absorbance ratio. 

Applications to fluorescent or fluorescently labeled proteins and nucleic acids, and to fluorescent lipid probes in phospholipid bilayers, have been reported. In the latter case, the diffusion coefficients measured above the chain melting temperature were found to be 10 7 cm2 s 1, which is in agreement with values obtained by other techniques. 

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

Fluorescently labeled proteins are compatible with mass spectrometry... 

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],... 

Microdialysis was achieved in a fused silica chip with in situ photopattemed porous membrane, as shown in Figure 5.13. Phase-separation polymerization of the membrane (7-50 pm thick) was formed between posts. The posts maximize the mechanical strength of the membrane so that it can withstand a pressure drop of 1 bar. Low MW cutoff (MWCO) membrane, which can be formed by using less organic solvent, 2-methoxyethanol, appears to be more transparent (see Figure 5.13). This low MWCO membrane can be used to dialyze away low MW molecules, such as rhodamine 560, but not fluorescently labeled proteins (insulin, BSA, anti-biotin, and lactalbumin). Fligh MWCO membrane, which was formed by more organic solvent, allows diffusion of lactalbumin [347]. 

TAS = 0) obtained with the 5 10°C temperature shift and the proportionally small optical changes in UV-Visible spectroscopy associated with the transient disequilibrium state. However, these limitations do not seem to apply to protein folding studies in which changes of tryptophan fluorescence or that of the fluorescently labeled proteins were monitored. In addition to a temperature jump, protein unfolding can also be rapidly initiated by a pressure jump. The dead time of the recently developed pressure-jump instrument is 50 ts for a pressure jump of 100 bar. ... 

Figure 3 Cross-correlation microscopy to study protein dynamics at the plasma membrane, (a) Methodology for analyzing protein dynamics at the plasma membrane. Cells are transiently transfected with the fluorescently labeled protein of interest. The antigen receptor and plasma membrane are also labeled with fluorescent markers. Plasma membrane masks generated for each time point are used to quantify protein dynamics at the plasma membrane over the time course. Representative traces of two cells that express GFP-PLCy-(SH2)2 showing (b) recmitment to the plasma membrane and (c) interactions with clustered IgE receptors after stimulation as described in Reference 61.

A new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labelled solute molecules at surfaces was developed by Thompson et al.74). The technique combines total internal reflection fluorescence with either fluorescence photobleaching recovery or fluorescence correlation spectroscopy with lasers. For example, fluorescent labelled protein was studied in regard to the surface chemistry of blood 75). 

Protein expression/localization/turnover and trafficking (or translocation) may be assessed by traditional immunofluorescence staining at various fixed time points, or in live cells by the use of fluorescently tagged proteins. The use of fluorescently labelled proteins, however, requires careful validations, as the tag may... 

Support-bound combinatorial Ubraries can be prepared with the chemical properties, as described earlier. Affinity methods were developed where soluble fluorescently labeled proteins were mixed with the library and noncovalently bound to the minority of beads containing active sequences. Using a flow cytometer, the beads with fluorescently bound protein can be sorted from the majority of beads that do not contain the active sequence and the compounds on the active beads sequenced by MALDI-MS. In a model study, an... 

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. 

An intriguing new development relates to conditional protein splicing [27]. In this scenario the property of the immunosuppressant rapamycin is utilized to bind simultaneously to two different proteins (FKBP and FRB) with high affinity. If FKBP and FRB are linked to the N- and C-termini of the intein-extein halves (see Fig. 4 for the protein constructs) the addition of rapamycin would trigger the formation of a ternary complex, thereby bringing the intein halves into close proximity. In a consecutive reaction the two exteins can combine to generate an active or, e.g., fluorescent labeled protein. Since both halves can be expressed in mammalian and other cells, cellular functions can be studied. 

Transport of a fluorescently labeled protein along the secretory pathway can be observed by microscopy (see Figure 17-2). Transport of a radiolabeled protein commonly is tracked by following compartment-specific covalent modifications to the protein. 

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. 

Fluorescent spectroscopy, because of its high level of sensitivity, has long been a powerful method for studying protein behavior. Site-specific attachment of fluorophores to a unique cysteine in a protein of interest is a traditional route for the production of fluorescent proteins. In addition, the discovery of fluorescent proteins, such as the green fluorescent protein (GFP) from the jellyfish Aequorea victoria [62], has provided a genetic approach for the production of fluorescently labeled proteins. Both these methods, however,... 

Fluorescent probes have been extremely useful in elucidating biochemical mechanisms and processes inside of living cells via fluorescent microscopy. This technique is particularly valuable because it is non-destructive and the probes can be observed in real time over the course of cellular events. Fluorescent probes fall into two main classes chemosensors and biosensors. Biosensors are fluorescently labelled proteins, most often antibodies. These types of probes have the disadvantage of poor cell permeability, but can be generated with specificity for any macromolecule against which an antibody can be raised. Chemical sensors are typically based on synthetic compounds and have been used in cells mainly to quantify the concentration of certain... 

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