Buffer system, discontinuous

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. 

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... 

The Tris-glycine discontinuous buffer system of Laemmli cannot be used for the separation of proteins with molecular weights < 10 to 15 kDa. For the analysis of smaller proteins, an alternative Tris-tricine buffer system is used along with an acrylamide solution that has a high percentage of cross-linker. This technique should be used when separating peptides in the size range of 1 to 20 kDa. 

Protein concentrations were determined according to the method of Lowry et al. (30). Electrophoresis of proteins in polyacrylamide gels was carried out at 4°C, using the discontinuous buffer system No. 1 described by Maurer (31) and modified by Emert et al. (1). Protein was stained with 0.1% Coomassie Brilliant Blue R250 in a water-acetic acid-methanol (45 10 45) solution. Carbohydrates were stained with the periodic acid-Schiff (PAS) reagent using the method described by Lang (32). 

S.J. Locke and P. Thibault, Improvement in detection limits for the determination of paralytic shellfish poisoning toxins in shellfish tissue using capillary electrophoresis electrospray mass spectrometry and discontinuous buffer systems, Anal. Chem., 66, 3436-3446 (1994). 

In our experience with cultivated and wild soybean tissues, a discontinuous buffer system using 5 mM L-histidine (pH 7.0) as the gel buffer and 0.13 M Tris-40 mM citrate (pH 7.0) as the electrode buffer works well for the 23 enzymes we routinely assay. We employ this single buffer system whenever possible so that we can assay for as many enzymes as possible on a single gel. 

Capillary isotachophoresis (CITP) — An electrophoretic separation technique (-> electrophoresis) in a discontinuous -> buffer system, in which the analytes migrate according to their -> electrophoretic mobilities, forming a chain of adjacent zones moving with equal velocity between two solutions, i.e., leading and terminating electrolyte, which bracket the mobility range of the analytes. Ref [i] Riekkola ML, Jonsson jA, Smith RM (2004) Pure Appl Chem 76 443... 

A discontinuous buffer system can be applied to increase the resolution and loadability of protein solutions. The application of larger volumes of the sample solution helps improve detectability of the system. The samples can be introduced by means of pressure injection, and after running the experiment, the gel matrix can be replaced by using pressure rinsing. Proteins of fairly high molecular weights can be separated by this approach. 

In discontinuous buffer systems especially (Davis 1964 Ornstein 1964), the conductivity of the gel varies through the run. For this reason constant-current power supplies are always advocated. Though the resistance always varies somewhat, probably largely while the gel warms up to its equilibrium running temperature, it is not our... 

Preparation of gels for discontinuous SDS-containing Tris buffer systems ... 

For the discontinuous buffer systems, the three widely used protocols, i.e., for high pH (Davis, 1964 Ornstein, 1964), neutral pH (Williams and Reisfeld, 1964) and low pH (Reisfeld et al., 1962), are in Table 16.5. The neutral buffer system has a poor buffering capacity and is only useful for proteins which are unstable in the high pH system. The low pH system is used for basic proteins (histones, several of the POase isozymes). It is imperative to check carefully the pH of all solutions. 

Experiments were attempted with both ethanol and manganous ion present. The results conformed to no simple rate law and seemed only understandable if it was assumed that the manganous ion (at <4 x 10-1 M) was being complexed by an impurity in the ethanol. Purification of the ethanol by distillation increased the rate a. 4 fold but did not eliminate the effect. Because of the apparent participation of the buffer in the rate law, work on buffered systems was discontinued. 

Standard discontinuous alkaline buffer system for polyacrylamide-gei electrophoresis of low-isoelectric-point proteins... 

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