Electrophoresis microscopic

Electrophoresis (qv), ie, the migration of small particles suspended in a polar Hquid in an electric field toward an electrode, is the best known effect. If a sample of the suspension is placed in a suitably designed ceU, with a d-c potential appHed across the ceU, and the particles are observed through a microscope, they can all be seen to move in one direction, toward one of the two electrodes. AH of the particles, regardless of their size, appear to move at the same velocity, as both the electrostatic force and resistance to particle motion depend on particle surface this velocity can be easily measured. 

Response to Electric and Acoustic Fields. If the stabilization of a suspension is primarily due to electrostatic repulsion, measurement of the zeta potential, can detect whether there is adequate electrostatic repulsion to overcome polarizabiUty attraction. A common guideline is that the dispersion should be stable if > 30 mV. In electrophoresis the appHed electric field is held constant and particle velocity is monitored using a microscope and video camera. In the electrosonic ampHtude technique the electric field is pulsed, and the sudden motion of the charged particles relative to their counterion atmospheres generates an acoustic pulse which can be related to the charge on the particles and the concentration of ions in solution (18). 

Norden, B Elvingson, C Jonsson, M Akerman, B, Microscopic Behavior of DNA Duing Electrophoresis Electrophoretic Orientation, Quarterly Reviews of Biophysics 24, 103,1991. Nozad, I Carbonell, RG Whitaker, S, Heat Conduction in Multiphase Systems—I Theory and Experiment for Two-Phase Systems, Chemical Engineering Science 40, 843, 1985. 

In this section, we shall first give a brief review of the phenomenological theory of these effects.5 -6 26 We shall then show how the methods we have discussed in the previous sections may be extended to derive a microscopic theory of the relaxation effect the microscopic theory of electrophoresis will be considered in the next section. 

VI. MICROSCOPIC THEORY OF ELECTROPHORESIS AN EXAMPLE OF HYDRODYNAMICAL LONG-RANGE... 

We are now in a position which allows us to give a microscopic analysis of electrophoresis. However, the detailed calculations rapidly become extremely complicated and we shall often be obliged to replace strict mathematical proofs by physical plausibility arguments. 

It is notable that the GC samples in Table 4.1 are much too short for the Intermediate Zone formula to apply out to 120 ns. The formulas for subsequent zones of C (t) (Eqs. 4.38—4.41) are employed as needed and yield the same value of a for both 230- and 590-bp samples.(146) The 590-bp sample initially exhibited a threefold higher value, which relaxed over several months, during which time many very small fragments dissociated from, or annealed out of, the predominant 590-bp species. This was tentatively attributed to the presence of branched structures, which exhibit high affinity sites for ethidiuny in the original material. Both gel electrophoretic and electron microscopic 147 1 evidence for branched structures in poly(dG-dC) were noted.(146) The 500-bp length from gel electrophoresis was confirmed by sedimentation.(146)... 

Visual examination of crystals using a light microscope does not indicate whether the crystals consist of only the protein or the protein-DNA complex. Therefore, the crystals are washed free of any uncrystallized DNA and protein several times with a solution containing the precipitant and any additives, etc. at the concentration and pH used for growing crystals (mother liquor). Finally, the crystals are separated from the mother liquor by microcentrifugation, dissolved in a suitable buffer, and analysed biochemically. The protein content is determined by SDS polyacrylamide gel electrophoresis, the protein concentration by BIO-RAD assay, and amino acid composition by mass-spectroscopy. The DNA can be detected by staining the gel with ethidium bromide or methylene blue (Jordan et al., 1985), whereas... 

The rate of particle migration is determined by measuring with a stopwatch the time required for a particle to travel between the marks of a calibrated graticule in the microscope eyepiece. If the objective of the microscope is immersed during the electrophoresis measurement, the calibration of the graticule should be made with the same immersion liquid. 

Another useful physical property of the crystal is its density, which can be used to determine several useful microscopic properties, including the protein molecular weight, the proteinlwater ratio in the crystal, and the number of protein molecules in each asymmetric unit (defined later). Molecular weights from crystal density are more accurate than those from electrophoresis or most other methods (except mass spectrometry) and are not affected by dissociation or aggregation of protein molecules. The proteinlwater ratio is used to clarify electron-density maps prior to interpretation (Chapter 7). If the unit cell is symmetric (Chapter 4), it can be subdivided into two or more equivalent parts called asymmetric units (the simplest unit cell contains, or in fact is, one asymmetric unit). For interpreting electron-density maps, it is helpful to know the number of protein molecules per asymmetric unit. 

Platinum films [27,88], microwires [19,84] and microdisks [12,103] were also employed. Characterisation of electrode fouling and surface regeneration for platinum electrode on an electrophoresis microchip was reported [104], The platinum tip of a scanning electron microscope has also been used for carrying out EC measurements combined in an end-configuration to a CE microchip [76]. 

Good descriptions of practical experimental techniques in conventional electrophoresis can be found in Refs. [81,253,259]. For the most part, these techniques are applied to suspensions and emulsions, rather than foams. Even for foams, an indirect way to obtain information about the potential at foam lamella interfaces is by bubble electrophoresis. In bubble microelectrophoresis the dispersed bubbles are viewed under a microscope and their electrophoretic velocity is measured taking the horizontal component of motion, since bubbles rapidly float upwards in the electrophoresis cells [260,261]. A variation on this technique is the spinning cylinder method, in which a bubble is held in a cylindrical cell that is spinning about its long axis (see [262] and p.163 in Ref. [44]). Other electrokinetic techniques, such as the measurement of sedimentation potential [263] have also been used. 

FIGURE 7.3 The rotary confocal fluorescence scanner used to detect pCAE chip separations. Laser excitation at 488 nm (100 mW) is directed up through the hollow shaft of a computer-controlled stepper motor, deflected 1.0 cm off-axis by a rhomb prism, and focused on the electrophoresis lanes by a microscope objective. The stepper motor rotates the rhomb/objective assembly just under the lower surface of the microchip at five revo-lutions/s. Fluorescence is collected along the same path and spectrally, and is spatially filtered before impinging on the four-color confocal detector [980]. Reprinted with permission from the American Chemical Society. 

Uchiyama, K., Hibara, A., Sato, K., Hisamoto, H., Tokeshi, M., Kitamori, T., An interface chip connection between capillary electrophoresis and thermal lens microscope. Electrophoresis 2003, 24, 179-184. 

Jiang, G., Attiya, S., Ocvirk, G., Lee, W.E., Harrison, D.J., Red diode laser induced fluorescence detection with a confocal microscope on a microchip for capillary electrophoresis. Biosens. Bioelectronics 2000, 14, 861-869. 

Forensic biochemists perform blood typing and enzyme tests on body fluids in cases involving assault, and also in paternity cases. Even tiny samples of blood, saliva, or semen may be separated by electrophoresis and subjected to enzymatic analysis. In the case of rape, traces of semen found on clothing or on the person become important evidence the composition of semen varies from person to person. Some individuals excrete enzymes such as acid phosphatase and other proteins that are seldom found outside seminal fluid, and these chemical substances are characteristic of their semen samples. The presence of semen may be shown by the microscopic analysis for the presence of spermatozoa or by a positive test for prostate specific antigen. 

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