Buffer subtraction

Figure 19.6 illustrates how we process the two-energy data and generate the ASAXS profiles. First, we average the set of all DNA and buffer intensity profiles at each of the two energies. The averaged data are displayed in Fig. 19.6A. Note that both DNA and buffer profiles, taken at Eon, have slighdy elevated background relative to Eofr data this is due to X-ray fluorescence. Most of this fluorescence background is removed by buffer subtraction (see Fig. 19.6B). The remaining on-edge fluorescence results...

Figure 19.6 Demonstration of data processing to generate ASAXS profiles. Panel (A) shows the raw data, acquired at the two ASAXS energies, along with the corresponding buffers. Panel (B) shows the buffer subtracted curves at each energy. Panel (C) shows the data after corrections have been applied to account for energy dependent effects and for fluorescence, detailed in the text. Panel (D) illustrates the measured difference signal I(Eoff) I...

After monodispersity tests, the most important physical assay is that of the sample and buffer transmission measurement. These are required for accurate sample-buffer subtractions (below) and for and matchpoint determinations (Section 2.5). Since the neutron transmissions of HjO and H20 range from about... 

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.

Each thermogram was normalized on scan rate, the corresponding (scan-rate-normalized) buffer-buffer baseline was subtracted, and the differential heat capacity values were divided by the number of moles of protein or peptide in the sample, to yield ordinate values in terms of calories moF deg. The resulting files were then analyzed using the deconvolution software. 

Selected entries from Methods in Enzymology [vol, page(s)] Aspartate transcarbamylase [assembly effects, 259, 624-625 buffer sensitivity, 259, 625 ligation effects, 259, 625 mutation effects, 259, 626] baseline estimation [effect on parameters, 240, 542-543, 548-549 importance of, 240, 540 polynomial interpolation, 240, 540-541,549, 567 proportional method for, 240, 541-542, 547-548, 567] baseline subtraction and partial molar heat capacity, 259, 151 changes in solvent accessible surface areas, 240, 519-520, 528 characterization of membrane phase transition, 250,... 

The measurement has to be done against tbe protein-free solvent (buffer). If a single-beam photometer is used, the absorbance of this blank has to be subtracted from that of the protein solution. 

Besides the enzymatic incubation in the reaction mixture, all procedures are carried out at 4°C. GTPCH activity is assayed by measuring the neopterin produced upon enzymatic incubation at 37°C for 60 min in a final volume of 0.1 ml in the dark (due to light sensitivity of pterins), followed by chemical oxidation and dephosphorylation. Two separate blanks are prepared, a blank reaction with cell lysate that is immediately oxidized to detect the neopterin that was present in the lysate, and a blank reaction without cell lysate to detect the neopterin that is generated from the incubation (substrate) buffer. The sum of both blanks is later subtracted from the value of the incubation reaction to determine the enzymatically produced neopterin. 

Figure 19-9 (a) Spectrophotometric titration of 30.0 mL of EDTA in acetate buffer with CuS04 in the same buffer. Upper curve [EDTA] = [Cu2 ] = 5.00 mM. Lower curve [EDTA] = [Cu2 ] = 2.50 mM. The absorbance has not been corrected in any way. (b) Transformation of data into mole fraction format. The absorbance of free CuS04 at the same formal concentration has been subtracted from each point in panel a. EDTA is transparent at this wavelength. [From L D. Hill and P MacCarthy, Novel Approach to Job s Method Chem. Ed. 1986,63, 162.]... 

To correct for possible fluorescent contamination in the RNAase solution, place 3.0 mL of pH 7.5, ethidium bromide-Tris buffer in a cuvette. Add 15 fiL of Tris buffer I and 2 fxL of ribonuclease. Mix well and incubate at 37°C for 20 minutes. Record the fluorescence intensity, if any. If the fluorescence is significant (> 1 intensity unit), subtract from Adna x before calculation of unknown DNA. Calculate the concentration of DNA in your sample in units of /ag/mL. Was RNA present in your unknown DNA solution ... 

Figure B3.5.8 Obtaining the corrected near-UV CD spectrum for hen egg white lysozyme. The protein and baseline spectra were collected using a 10-mm cylindrical cell and 0.5 mg/ml protein in 0.067 M phosphate buffer, pH 6.0. Instrument settings were 1-nm bandwidth, 0.2-nm step size, scan speed 2 nm/min, time constant 8 sec (scan speed x time constant = 0.27 nm). Protein solution and buffer were scanned once each. The spectra were smoothed, a sample of the fit being shown in the inset. Reproducibility of the instrument and of the state of the cell are demonstrated by the coincidence of the ellipticity above 300 nm. The corrected spectrum was obtained by subtraction, using the instrument software.

A further problem that is sometimes encountered is that the spectrum obtained after baseline subtraction does not return to zero above 320 nm (in near-UV CD) or 250 nm (in far-UV CD). If, above these wavelengths, the spectrum runs parallel to the zero base, a provisional spectrum may be derived by subtracting the difference in ellipticity, as a constant, from the whole spectrum. For quantitative assessment of a sample, however, the spectrum should be redetermined, paying attention to the correct buffer blank, cell reproducibility, and instrumental drift (see Strategic Planning). 

Apart from the Raman band, a buffer solution should give an almost flat baseline of low intensity (see Fig. B3.6.I). At the usual working concentrations of protein (A280 >0.05), buffer fluorescence constitutes only a minor contribution to the fluorescence spectrum of the protein and is accounted for by the appropriate baseline subtraction. 

Perform a control assay by filling the cell with 25°C buffer and injecting an identical amount of enzyme but no substrate. Subtract the rate of the control from that of the assay containing substrate. 

When a determination is critical, it is often necessary to do two or more different colorimetric tests in parallel. A substance present in the sample could interfere with one test while not affecting others. In most cases, interference can be controlled. Interference can be detected by adding a known amount of sugar to a sample solution to be measured. The amount of sugar will differ from what is expected if there is interference. In addition, a very colored sample can interfere with the results obtained. In the case of colored samples, subtract the absorbance of the samples dissolved in water or buffer alone from the value obtained after addition of the colorimetric reagents. 

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