Cellulose acetate electrophoresis of casein proteins.

Samples of the milk proteins α_s1-casein and β-casein partially dephosphorylated by means of bovine spleen phosphoprotein phosphatase have been electrophoretically analysed using cellulose acetate as the supporting medium and Procion blue as the protein dye. Sufficient resolution was obtained in 1 hr to allow quantification of the proteins present. Skimmed-milk samples and acid-precipitated whole casein samples have been analysed by the same technique. The advantages of the method are discussed in relation to the more conventional electrophoretic techniques normally used to analyse these milk proteins.     

The use of liquid polyacrylamide in electrophoresis: III. Properties of liquid polyacrylamide in the presence of cellulose acetate

Solutions of uncross-linked liquid polyacrylamide (LPA) stabilized by cellulose acetate are shown to represent efficient molecular sieves for RNA and for sodium dodecyl sulfate-protein complexes. Endosmosis and evaporation have to be considered to obtain corrected estimates of mobilities of the macro-ions. The efficiency of molecular sieving is strongly influenced by the chain length of the single polymer molecules. This can be shown both in terms of the steepness of the relationship between logarithm of molecular weight and mobility and in the relationship between log (K_R) and log molecular weight. Comparable chain length effects are found in cross-linked polyacrylamide, suggesting a common mechanism of action of the two electrophoretic sieving media. The correlation of polymer viscosity and of the impedance towards migration of macro-ions, as demonstrated by LPA-electrophoresis, suggests the participation of hydrodynamic interactions in the molecular sieving process.     

Cellulose acetate electrophoresis of protein kinases: Detection of the active forms using various substrates

Electrophoresis on cellulose acetate strips was used to analyze protein kinases from normal rat liver. In addition to already well-characterized cAMP-dependent protein kinases type I and II and cAMP-independent casein kinases I and II, this method enabled the detection of several supplementary bands corresponding to kinases which were investigated according to their substrate specificity, activation by cAMP, and inhibition by the specific inhibitor of the catalytic subunit of cAMP-dependent protein kinases or by heparin. Using this rapid, sensitive, and resolutive electrophoretic method, different isozyme patterns could be obtained starting from minute amounts of different types of biological material.     

Cellulose acetate membrane electrophoresis and FTIR spectroscopy as methods of identifying a fucoidan in Fucusvesiculosus Linnaeus

A fucoidan from brown algae such as the common bladder wrack (Fucusvesiculosus Linnaeus) is now widely examined in many countries for its interesting biological and therapeutic properties. In this study, the fucoidan was identified during extraction in hydrochloric acid; its presence was confirmed by FTIR spectroscopy. Two slightly different structures were found in real samples of dried bladder wrack supplied by Flos and Witherba, by comparing them with a reference sample of F. vesiculosus L. A simple, repeatable analytical procedure was developed using apparatus for cellulose acetate membrane electrophoresis and this was supplemented by semi-quantitative analysis.     

Differentiation of mite species by using cellulose acetate and equilibrium polyacrylamide gel electrophoresis of isoenzymes

The isoenzymes of various species of medically and economically important mites were studied using cellulose acetate and equilibrium polyacrylamide gel electrophoresis. Interspecific differences in isoenzymes were found, which were species-specific. Intraspecific differences in isoenzymes were also found when individual mites were examined. The efficiency of each technique, and their use in various fields of acarology, are discussed. In addition, possible phylogenetic relationships as revealed by these techniques are suggested.     

Visible labeling of proteins for polyacrylamide gel electrophoresis with dabsyl chloride

Several proteins, which are used as molecular weight markers in polyacrylamide gel electrophoresis, were reacted with dabsyl chloride. This labeled them deep orange and the chromophore attachment was stable throughout the electrophoretic procedure and fixation. Small amounts (10-50 micrograms) of the labeled proteins could be loaded onto gels and seen with the unaided eye so that the separation during electrophoresis could be followed. Dabsylation did not affect the mobility of the proteins. The location of the orange band gave a good indication of the position of the protein in the gel so that molecular weight estimations could be made during and immediately following electrophoresis.     

An exponential gradient marker for use with minigel polyacrylamide electrophoresis systems

Minigel electrophoresis has reduced the required sample size and cost of running polyacrylamide gels without loss of resolution. The many problems incurred in the minigel system when pouring multiple gradient gels are eliminated by pouring individual gels with the described economical, exponential gradient marker, which can accurately deal with the small gel volumes. The advantages of exponential gradients are discussed.     

A method for horizontal polyacrylamide slab gel electrophoresis

We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.     

Apparatus for discontinuous electrophoresis in polyacrylamide gel slabs

Construction of an inexpensive slab discontinuous electrophoresis apparatus is described. Using this apparatus 23 human serum proteins were resolved and the gel could be scanned with a standard densitometer to yield a trace with discrete peaks or pronounced shoulders for each protein band. The advantages of a single homogeneous slab for comparative studies are indicated.     

A vertical submarine electrophoresis apparatus for polyacrylamide minigels

A vertical submarine electrophoresis apparatus for use with minislab polyacrylamide gels is described. The design allows polyacrylamide gels to be run with the same ease and convenience that agarose gels are run with horizontal submarine apparatuses. The vertical submarine features a single buffer chamber with a restriction between the upper and the lower portions of the chamber. Acrylamide gels, cast between 9 X 10-cm glass slides, are inserted into the restriction and are completely immersed in buffer. Thus, current flows primarily through the gel itself, but some current flows through the buffer in the restriction surrounding the gel. Because water-tight separation of buffer chambers is not necessary, time-consuming and/or expensive procedures such as sealing with agarose or using fragile notched glass plates are eliminated. The apparatus can be set up to run a gel in less than 30 s. It is versatile in that gels of varying thickness (0.5, 0.8, 1.5, and 3 mm) can be run on a single apparatus. The apparatus has been used for sodium dodecyl sulfate gels, low ionic strength native gels for nucleoprotein complexes, and composite acrylamide-agarose gels.