Electrophoresis experimental conditions

The nonquantitative detection of radioactive emission often is required for special experimental conditions. Autoradiography, which is the exposure of photographic film to radioactive emissions, is a commonly used technique for locating radiotracers on thin-layer chromatographs, electrophoresis gels, tissue mounted on sHdes, whole-body animal sHces, and specialized membranes (13). After exposure to the radiolabeled emitters, dark or black spots or bands appear as the film develops. This technique is especially useful for tritium detection but is also widely used for P, P, and 1. 

The use of standards with samples makes zone electrophoresis particulady usehil as an analytical tool. However, when samples caimot be analyzed on the same gel, differences in the experimental conditions from experiment to experiment make direct comparison more difficult. To make comparisons from experiment to experiment, a relative mobility, is often measured by measuring the distance a component travels down the gel compared to some reference or standard component. 

Analytical methods are ripe for attack using Al methods. Capillary electrophoresis is a routine separation technique, but like other separation techniques, its effectiveness is correlated strongly with experimental conditions. Hence it is important to optimize experimental conditions to achieve the maximum degree of separation. Zhang and co-workers41 studied the separation of mixtures in reserpine tablets, in which vitamin B1 and dibazolum may be incompletely separated, as may promethazine hydrochloride and chloroquine... 

Fig. 3.161. (A) Zone electrophoresis patterns of FITC-labelled transferrin samples by fluorescence detection. The unbound dye (providing a main peak and several minor ones) was not removed from the samples. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length 41 cm) X 75 pm i.d. injection of samples 100 mbar x s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut-off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 13 pm (1 mg/ml) Tf and (a) 0.01 mM FITC, (b) 0.1 mM FITC, and 1 mM FITC. (B) Zone electrophoresis patterns of an FITC-labelled transferrin sample by simultaneous fluorescence (upper trace, left axis) and UV detection (lower trace, right axis). The unbound dye shows several peaks with both detections. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length fluorescence 41 cm, UV 50.5 cm) X 75 pm i.d. injection of samples 100 mbar X s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 6.5 pm (0.5 mg/ml) Tf and 0.1 mM FITC. Reprinted with permission from T. Konecsni et al. [199].

Fig. 3.172. Non-aqueous capillary electrophoresis with electrochemical detection of a dye mixture containing (a) 1.7 jUg/ml malachite green, (b) 0.70 jug/ml crystal violet, (c) 4.3 /ig/ml rhodamine B, and (d) 9.1 X 10-6 M ferrocene. Experimental conditions capillary dimensions, 95 cm X 75 pm i.d. running electrolyte, acetonitrile containing 1 M HAc and 10 mM NaAc electrokinetic injection, 20 s 5 kV separation voltage 20 kV applied detection potential, 1.55 V. Reprinted with permission from F.-M. Matysik [206].

FIGURE I I Capillary zone electrophoresis of forced degraded rMAb sample. Experimental conditions as in Figure 9. 

This equation was derived above for the movement of a liquid through a stationary solid phase. Its application here to the movement of colloidal particles under experimental conditions that render the liquid medium immobile implies that the solid particle is large compared with the dimensions of the diffuse double layer k 1. It is customary to term this movement of the solid phase electrophoresis. The phenomenon is observed with particles suspended in a liquid (Fig. 6.139). 

One variant of absorbance detection that is widely used in HPLC can also be used in high performance capillary electrophoresis. For compounds that exhibit a very weak UV absorption, buffers such as chromate or phthalate, which have high absorption properties, can be used. Under these experimental conditions, the UV absorbance diminishes as analytes flow past the detector (due to the dilution effect of the electrolyte). This leads to negative peaks on the recorder (see Fig. 8.9). 

Before examining contemporary proteomic workflows, it must be said that the older gel-based technology still remains the largest contributor of proteomic-level information. For many years, the main means to analyze complex protein samples involves separation of proteins on the basis of size and isoelectric point (pi) using two-dimensional polyacrylamide gel electrophoresis (2-DE). The densitometric aspects of gel protein spots provide a direct means to visualize and quantify proteins based on the experimental conditions applied to the... 

Capillary electrophoresis (CE) provides a valid alternative to HPLC methods for chiral separations. The direct resolution of racemates requires only an enantiomerically pure additive (chiral selector) to be dissolved in the running buffer. The experimental conditions affecting the separations and an overview of practical applications have been compiled <1999ELP2605>. 

Electrophoresis in a mixed solvent at different temperatures was performed using the device described in Section III with the following experimental conditions. All stock solutions were prepared in aqueous medium, ethylene glycol (distilled under reduced pressure and kept at +4°C in the dark) being added just before the experiment to give a final volume ratio of 50%. This procedure led to more reproducible results. 

Figure 4. 2-D Gel Electrophoresis of in vitro Translated Soybean Epicotyl mRNA. Experimental conditions are as described in Figure 3. B = epicotyl sections auxin depleted for 2 hours followed by incubation with 5 x 10"5 M 2,4-D for 2 hours B = epicotyl sections auxin depleted as in B followed by incubation with 3.4 x 10 7 M BR for 30 minutes with a further treatment of both 3.4 x 10-7 M BR and 5 x 10 5 M 2,4-D for 2 hours.

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