Container/Buffer Experiment

If the secretory product is rather lipophilic, add a small amount of BSA-containing buffer to the Eppendorf tubes to which the supernatants are transferred at the end of the experiment. 

In a sensor experiment (e.g., in a typical biochemical experiment) the sensor has been functionalized with a sensitive film, which is in contact with an analyte containing buffer solution. The new resonance frequencies / can be calculated when replacing Ls and Rs by =Ls + Lc + Liiq and R = Rs + Knq, respectively (neglecting Qiq and Guq). Usually frequency shifts are determined and of interest only. Some example data are added to Table 2. Series and parallel resonance frequency shifts vary by a few percent. All the parallel resonance frequencies are very much affected by external capacitance (values in brackets). The same holds for all frequency shifts in a Hquid except /s. Oscillators based on parallel resonance should not be used because stray capacitance is hardly to avoid and hardly to keep constant in an experimental setup. Deviations of/r and/m from s are also ampHfied by external capacitance. 

Figure 1.2 The classical continuous labeling (bottom-up) HX-MS experiment. HX of an equilibrated protein solution is initiated by dilution into a Dfl-containing buffer, and exchange is quenched at various time points. Global HX (protein level) can be measured directly by liquid chromatography (LC) and mass spectrometry (MS) analysis of the intact protein, or local HX (peptide level) can be measured by enzymatic cleavage and subsequent LC-MS analysis of the proteolytic peptides. (See insert for color representation of the figure.)...

Figure 5.25 Plot of formal potential ( ° = (Epa — pc)/2) vspHforpoly(3-methyl-pyrrole-4-carboxylic acid) in 1M KNO3 containing 0.2M acetate buffer ( ). Experiments in the pH range 6.5-9.5 were repeated with 0.1 M NH4NO3 added to the cell solution (X). (Reprinted from Journal of Electroanalytical Chemistry, 225, P. G. Pickup, 273. Copyright (1987), with permission from Elsevier.)...

Adsorption of PLL-g-PEG as well as all in situ experiments and protein studies was conducted in a 4-(2-hydroxyethyl)-piperazine-l-ethane-snUbnic acid- (HEPES-) based buffer (Flu-ka, Buchs, Switzerland) adjusted to pH 7.4 with NaOH 6 M (Fluka, Buchs, Switzerland). The choice of the buffer system is important for the type of studies performed in this work While phosphate-containing buffers result in strong binding of phosphate to transition metal cations and therefore change the surface properties in a time-dependent manner, HEPES is a buffer system with only weak interaction with metal oxide surfaces. The ionic strength of the buffer varied between 1 and 160 mM, either by dilution or by addition of NaCl (Fluka, Buchs, Switzerland). The following buffers were prepared 1... 

From the data in Fig. 4.8b, estimate the shift factors required to displace the data at 0 = 0.5 (consider only this point) so that all runs superimpose on the experiment conducted at 128 C at 0 = 0.5. Either a ruler or proportional dividers can be used to measure displacements. Criticize or defend the following proposition Whether a buffered aqueous solution of H2O2 and 1. containing small amounts of S2O3 and starch, appears blue or colorless depends on both the time and the temperature. This standard general chemistry experiment could be used to demonstrate the equivalency of time and temperature. The pertinent reactions for the iodine clock are... 

Fig. 1.14 Relationship between the incorporation of 180 into product CO2 and the amount of luciferin used, in the bioluminescence reaction of Cypridina luciferin catalyzed by Cypridina luciferase. The reactions were carried out in H2160 medium with 1802 gas (solid line) in H2lsO medium with 16C>2 gas (dashed line) and the control experiment of the latter using C16C>2 gas instead of luciferin and luciferase (dotted line), all in 20 mM glycylglycine buffer, pH 7.8, containing 40 mM NaCl. From Shimomura and Johnson, 1973a. Reproduced with permission from Elsevier.

Why does EDTA cause only 90% inhibition, leaving 10% of the activity intact Buffer solutions usually contain 0.1 1 pM of contaminating Ca2+ when special precaution is not taken, and this concentration is much greater than the molar concentration of luciferase used in the experiments. Thus, one of the possibilities would be that Ca2+ interacts with the molecule of luciferase and can increase the activity of luciferase about 10 times, in spite of the fact that the molecule of luciferase lacks the Ca2+ binding site of EF-hand type (Thompson et al., 1989). Another possibility would be that EDTA interacts directly with the molecules of luciferase, to cause the inhibition. The question remains unresolved. 

Contaminating CO2. The ubiquitous presence of CO2 causes a great difficulty to the experiment. Normal atmosphere contains 0.03% CO2, and a 5-6 ml portion of pure water equilibrated with atmosphere contains 0.06 pmol of CO2. However, the amount of CO2 in buffers and luciferase solutions is much greater. For example, 5 ml of freshly prepared Tris-HCl buffer, pH 7.8, contained 0.13 pmol of CO2 after 20 min degassing, and the value increased to 0.23 pmol when the same buffer was tested one week later (Shimomura et al., 1977). It is strongly advised to eliminate all nonessential CO2 sources from the experimental environment. 

Cellulase and all chemicals used in this work were obtained from Sigma. Hydrolysis experiments were conducted by adding a fixed amount of 2 x 2 mm oflSce paper to flasks containing cellulase in 0.05 M acetate buffer (pH = 4.8). The flasks were placed in an incubator-shaker maintained at 50 °C and 100 rpm. A Box-Behnken design was used to assess the influence of four factors on the extent of sugar production. The four factors examined were (i) reaction time (h), (ii) enzyme to paper mass ratio (%), (iii) amount of surfactant added (Tween 80, g/L), and (iv) paper pretreatment condition (phosphoric add concentration, g/L), as shown in Table 1. Each factor is coded according to the equation... 

Whole cell OPH activity was measured by following the increase in absorbancy of p-nitrophenol from the hydrolysis of substrate (0.1 mM Paraoxon) at 400 nm (sm = 17,000 M cm ). Samples of culture (1 ml) were centrifuged at 10,000 g and 4 C for 5 min. The cells were washed, resuspended with distilled water, and 100 pi was added to an assay mixture containing 400 pi 250 mM CHES [2-(N-cyclohexylamino)ethane-sulfonic acid] buffer, pH 9.0, 100 pi 1 mM Paraoxon, and 400 pi distilled water. One unit of OPH activity was defined as pmoles Paraoxon hydrolyzed per min. Each value and error bar represents the mean of two independent experiments and its standard deviation. 

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