Fluorescence subtraction

Figure 2 Scatchard plot of TNP-ATP binding. Y= F-Fq/ F ax where Fq is the background fluorescence of TNP-ATP in buffer in the absence of protein and F is the observed fluorescence of TNP-ATP with protein fluorescence subtracted. X is the total iM concentration of TNP-ATP added.

One approach is to prepare a sample blank using urine known to be free of quinine. The fluorescent signal for the sample blank is subtracted from the urine sample s measured fluorescence. 

Kinetic data for total and nonspecific binding are subtracted to give the specific amount bound at the time of antibody addition, and the slow decline in fluorescence intensity thereafter reflects dissociation of bound peptide (also see Ref. 8 for more details). 

Figure 8. Simultaneous measurement of intracellular Ca and oxidant production in neutrophils. Cells were labeled with Quin-2 and suspended at 2 x lo cells/mL buffer. At time zero, 1 nJf FLPEP was added (upper trace in each panel). In addition, the receptor blocker tBOC was added (3 x 10" M) after 30 s to stop further binding of the stimulus (lower trace in each panel). The excitation wavelength was 3A0 nm. Top panel Quin-2 fluorescence determined on channel B (of Figure 1) using a Corion A90-nm interference filter. The crossover from the superoxide assay has been subtracted. Middle panel Oxidant production (superoxide equivalents) determined by the para-hydroxyphenylacetate assay. Fluorescence was observed at AOO nm (on channel A of Figure 1).

Fig. 8. Dependence of (A) corrected diffusion coefficient (D), (B) steady-state fluorescence intensity, and (C) corrected number of particles in the observation volume (N) of Alexa488-coupled IFABP with urea concentration. The diffusion coefficient and number of particles data shown here are corrected for the effect of viscosity and refractive indices of the urea solutions as described in text. For steady-state fluorescence data the protein was excited at 488 nm using a PTI Alphascan fluorometer (Photon Technology International, South Brunswick, New Jersey). Emission spectra at different urea concentrations were recorded between 500 and 600 nm. A baseline control containing only buffer was subtracted from each spectrum. The area of the corrected spectrum was then plotted against denaturant concentrations to obtain the unfolding transition of the protein. Urea data monitored by steady-state fluorescence were fitted to a simple two-state model. Other experimental conditions are the same as in Figure 6.

Other techniques that have been used include subtractive differential pulse voltammetry at twin gold electrodes [492], anodic stripping voltammetry using glassy-carbon electrodes [495,496], X-ray fluorescence analysis [493], and neutron activation analysis [494],... 

Various solutions have been proposed for the reduction or elimination of autofluorescence. One way is to chemically suppress the autofluorescence signal with some reagents such as sodium borohydride, glycine or toluidine blue. However, in many cases, these approaches are either infeasible or ineffective, and none of them fully eliminates the problem. The second way is to use spectral unmixing algorithms subtracting the background fluorescence. This is only possible if you have at your disposal complicated, expensive confocal optics with sophisticated automated software (http //www.cri-inc.com/applications/fluorescence-microscopy.asp). 

Subtraction of the acceptor fluorescence is possible but would decrease the accuracy if it is large. 

When plotting the variations in absorbance or fluorescence intensity versus x, it is convenient to subtract the absorbance or fluorescence intensity that would be measured in the absence of cation at every concentration, i.e. Y0(1 x). In this... 

In addition to a fluorescence perturbation, the Cd(II)-5d combination also uniquely yields aperturbation in the ultraviolet (UV) spectrum. A difference spectrum obtained by subtracting a fractional amount of an uncomplexed 5d spectrum from the perturbed spectrum is the mirror image of a fluorescence difference spectrum obtained by similar means. Moreover, excitation at 400 nm (where 1-4 are weakly absorbing but where moderate absorption is seen in the difference spectrum) gives rise to an emission spectrum with identical shape and Amax (456 nm) to that of the fluorescence difference spectrum. Thus, evidence points to the existence of two equilibrating ground state species as the physical basis for the chelatoselective emission. Bouas-Laurent has reported a related observation in methanol where a red-shifted CHEF was observed for a T1(I) 7r-complex.(14)... 

The mathematical treatment of each of the fluorescence curves shown in O Figure 5-5 is identical. First nonspecific effects must be subtracted so that in each case we have a relationship between the concentration of ligand and the change in fluorescence. The binding equation is formally identical to the terminology for... 

When fluorochromes are combined in a multiparameter assay, it is almost always necessary to correct for signals from overlapping portions of emission spectra that have not been eliminated by optical filtration. This is accomplished through a spectral compensation procedure that is performed according to instrument-specific instructions, and involves the adjustment of detector voltages to electronically subtract extraneous fluorescence. 

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