Buffer discontinuity

Assays. Protein concentrations were measured by the method of Bradford (18) and the various contractile protein ATPase activities by tRe method of Martin and Doty (19). Gel electrophoresis was carried out by the method of Ames (20) on 1.5 ran polyacrylamide slabs using the discontinuous SDS buffer system of Laemnli (21). Dried gels were scanned at 550 nm for densiometry measurements. 

Equations 2.58, 2.70 and 2.71 enable the velocity distribution to be calculated for steady fully developed turbulent flow. These equations are only approximate and lead to a discontinuity of the gradient at y+ = 30, which is where equations 2.70 and 2.71 intersect. The actual profile is, of course, smooth and the transition from the buffer zone to the fully turbulent outer zone is particularly gradual. As a result it is somewhat arbitrary where the limit of the buffer zone is taken often the value y+ = 70 rather than j + = 30 is used. The ability to represent the velocity profile in most turbulent boundary layers by the same v+ - y+ relationships (equations 2.58, 2.70 and 2.71) is the reason for calling this the universal velocity profile. The use of in defining v+ and y+ demonstrates the fundamental importance of the wall shear stress. 

Strambini and Galley have used tryptophan anisotropy to measure the rotation of proteins in glassy solvents as a function of temperature. They found that the anisotropy of tryptophan phosphorescence reflected the size of globular proteins in glycerol buffer in the temperature range -90 to -70°C.(84 85) Tryptophan phosphorescence of erythrocyte ghosts depolarized discontinuously as a function of temperature. These authors interpreted the complex temperature dependence to indicate protein-protein interactions in the membrane. 

The electrical current in an electrophoresis cell is carried largely by the ions supplied by buffer compounds. Proteins constitute only a small proportion of the current-carrying ions in an electrophoresis cell. Buffer systems for electrophoresis are classified as either continuous or discontinuous, depending on whether one or more buffers are used. They are further classified as native or denaturing, depending on whether their compositions maintain or destroy protein structure and activity. 

For detailed descriptions of the electrochemical processes that operate with discontinuous buffer systems, consult References 1, 2, 4-7, 13, and 20. Mathematically inclined readers might want to follow the development of multiphasic buffer theory as presented in References 21 to 23. 

The choice of native electrophoresis system depends on the particular proteins of interest. There is no universal buffer system ideal for the electrophoresis of all native proteins. Both protein stability and resolution are important considerations in buffer selection. Recommended choices are the Omstein-Davis discontinuous system21-24 and McLellan s continuous buffers.25... 

Ornstein21 and Davis24 developed the first high-resolution PAGE system for native proteins. Their popular system is still in widespread use. It was designed for the analysis of serum proteins, but works well for a broad range of protein types. The Ornstein-Davis buffers should be the first discontinuous system tried when working with a new, native sample. 

The choice of proper gel concentration (%T) is, of course, critical to the success of the separation because it heavily influences separation. Too high %T can lead to exclusion of proteins from the gel, and too low %T can decrease sieving (see Figure 8.4). One approach, useful with the McLellan continuous buffers (Table 8.1), is to use relatively large-pore gels (6%T or 7%T) and to alter mobilities with pH. An approach for discontinuous systems is to start with a... 

It is important to bear in mind that an electrophoresis gel is an element in an electrical circuit and as such obeys the fundamental laws of electricity. Each gel has an intrinsic resistance, R, determined by the ionic strength of its buffer (R changes with time in discontinuous systems). When a voltage V is impressed across the gel, a current I flows through the gel and the external circuitry. Ohm s law relates these three quantities V = IR, where V is expressed in volts, I in amperes, and R in ohms. In addition, power P, in watts, is given by P = IV. The generation of Joule heat, H, is related to power by the mechanical equivalent of heat, 4.18 J/cal, so that H = (PI4.18) cal/sec. 

Membranes were pre-compacted at 800 psig for approximately one hour, the pressure then being reduced to 600 psig for collection of performance data. At the beginning of this work, control samples were soaked in buffer solutions for times corresponding to membrane exposure. It was subsequently found that control samples were unaffected by buffer solution alone and this procedure was discontinued. 

Based on data with buffered didanosine formulations, patients with symptoms of peripheral neuropathy may tolerate a reduced dose after resolution of these symptoms following drug discontinuation. If neuropathy recurs after resumption of didanosine, consider permanent discontinuation. 

Another problem, the potential increase in background noise due to the addition of solvents and modifiers from the sheath liquid (e.g., volatile salts, acids, and bases), has been studied (19). Moreover, because of the different composition of the initial CE buffer reservoir and the sheath liquid, discontinuous and irreproducible conditions may result. These effects can potentially change migration times or even the migration order of the analytes (20). 

The protein release has discontinuous character and occurs between 22 °C and 32 °C in phosphate buffer. The latter is probably due to charge effects of proteins. 

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