实验仪器使用技术

Fluorescence recovery after photobleaching (FRAP) is one of the most useful microscopy techniques for studying the mobility of molecules in terms of a diffusion coefficient. Here, we describe a FRAP method that allows such measurements, relying on the photobleaching of a rectangular re ...

Environment-sensitive fluorescent membrane probes are attractive tools for investigating the membrane properties and their changes under perturbing conditions. Membrane probes of the 3-hydroxyflavone family are of particular interest due to their excited-state intra ...

Nowadays, fluorescent protein (FP) variants have been engineered to fluoresce in all different colors; to display photoswitchable, or photochromic, behavior; or to show yet other beneficial properties that enable or enhance a still growing set of new fluorescence spectroscopy and mi ...

Biosynthetic incorporation of Trp analogs in a protein can help in its characterization using fluorescence spectroscopy and other methodologies like NMR and phosphorescence. Here a protocol is presented resulting in the efficient incorporation of Trp analogs in a recombinant pr ...

Instantaneous fluorescence emission spectra measured at different times after excitation often shift to the red as the delay between the excitation pulse and fluorescence detection is increased. In the case of Trp fluorescence in proteins, the time-dependent red shift (TDRS) may have i ...

Principles behind quenching of tryptophan (Trp) fluorescence are updated and extended in light of recent 100-ns and 1-μs molecular dynamics (MD) trajectories augmented with quantum mechanical (QM) calculations that consider electrostatic contributions to wavelength shif ...

Most proteins are nanomachines that are selected to execute specific functions and therefore should have some degree of flexibility. The driving force that excites specific motions of domains and smaller chain elements is the thermal fluctuations of the solvent bath which are channel ...

Measuring changes in a molecule’s fluorescence emission is a common technique to study complex biological systems such as cells and tissues. Although the steady-state fluorescence intensity is frequently used, measuring the average amount of time that a molecule spends in the excited s ...

Frequency domain fluorometry is a widely utilized tool in the physical, chemical, and biological sciences. This chapter focuses on the theory of the method and the practical aspects required to carry out intensity decay, i.e., lifetime measurements on a modern frequency domain fluoromet ...

A method of analysis is described on the photoinduced electron transfer (PET) from aromatic amino acids as tryptophans (Trp) and tyrosines (Tyr) to the excited isoalloxazine (Isundefined) in FMN-binding proteins (FBP) from Desulfovibrio vulgaris (strain, Miyazaki F). Time-dependent geome ...

This chapter describes an experimental layout for time and spectrally resolved fluorescence measurements with femtosecond time resolution based on Kerr gating. The combination of data recorded using different Kerr media allows a temporal dynamic range from ~100fs to several nan ...

As the other chapters attest, sensitivity of fluorescent molecules to their local environment has created powerful tools in the study of molecular biology, particularly in the study of protein, DNA, and lipid dynamics. Surprisingly, even events faster than the nanosecond lifetimes of fl ...

Methods and protocols are described when using fluorescence metrology to determine the average nanoparticle (np) size in colloids in the range of 1–10nm. The technique is based on determining the rotational correlation time of the np from the decay of fluorescence anisotropy of a dye that is el ...

In this chapter, we describe the global analysis approach for processing time-resolved fluorescence spectroscopy data of molecules in the condensed phase. Combining simultaneous analysis of data measured under different experimental conditions (spatial coordinates, te ...

Fluorescence is the most widely used technique to study the effect of pressure on biochemical systems. The use of pressure as a physical variable sheds light into volumetric characteristics of reactions. Here we focus on the effect of pressure on protein solutions using a simple unfolding exa ...

This chapter describes a procedure of global analysis of the steady-state spectra measured with different concentrations of the denaturant to quantitatively study protein denaturation. With the help of physicochemical models, relevant spectral parameters that character ...

Fluorescence methods are often employed for the characterization of molecular interactions. In particular, polarization/anisotropy studies are widely utilized in the life sciences as they allow quantification of protein interactions in the micro- and nanomolar concentra ...

Contemporary spectrofluorimeters comprise exciting light sources, excitation and emission monochromators, and detectors that without correction yield data not conforming to an ideal spectral response. The correction of the spectral properties of the exciting and emissi ...

D166V point mutation in the ventricular myosin regulatory light chain (RLC) is one of the causes of familial hypertrophic cardiomyopathy (FHC). We show here that the rates of cross-bridge attachment and dissociation are significantly different in isometrically contracting cardi ...

Atomic force microscopy in its single-molecule force spectroscopy mode is a nanomanipulation technique that is extensively used for the study of the mechanical properties of proteins. It is particularly suited to examine their response to stretching (i.e., molecular elasticity and ...