Isoelectric focusing of horse acidic prealbumins on thin-layer polyacrylamide gels

The paper describes a technique of thin-layer polyacrylamide gel isolectric focusing of horse serum, within a pH range of 4.0 - 6.0, which permits the improved resolution of the acidic prealbumin protein bands. The increased heterogeneity of the Pr prealbumin antiprotease allele products apparent using this technique is described and discussed in detail, and the potential use of the technique in routine Pr phenotyping is considered.     

Isoelectric focusing of human granulocyte lysosomal elastase in thin-layer polyacrylamide gels

A technique is described which allows isoelectric focusing of human granulocyte elastase in thin-layer polyacrylamide gels, with retention of between 70 and 90% of the elastolytic activity. Digestion of elastin was observed to occur between pH 8.13 and 8.3 as well as between pH 8.77 and 9.15. Detection of elastolytic activity and visualization of protein bands following staining of the gels, has been successfully attempted with as little as 20 μg of lysosomal extract (about 2 to 3 μg of elastase).     

Isoelectric focusing in polyacrylamide-agarose

A procedure using a 2.5% acrylamide-0.5% agarose gel for slab or tube isoelectric focusing is described. This composite gel is durable and enables a rapid focusing of high-molecular-weight compounds.     

Isoelectric focusing of carboxypeptidase N.

Carboxypeptidase N was partially purified on a TEAE-cellulose column and subjected to isoelectric focusing in sucrose gradient columns containing ampholine gradients of pH range 3-10 and 4-8. Activity separated into two major peaks with pI values of pH 3.8 and 4.3. Both peaks were totally converted to an active desialated enzyme with isoelectric point of pH 5.2 to 5.4. These results indicate that carboxypeptidase N is a sialoprotein with at least two forms, differing in sialic acid content, in serum. Catalytic activity is not dependent upon sialic acid but the latter may possibly influence stability since loss of activity occurred in the desialated enzyme with repeat focusing.     

Isoelectric focusing of mycoplasma proteins.

Polyacrylamide gel isoelectric focusing (PAGIF) in thin layer was used to resolve proteins of Mycoplasma spp., Acholeplasma spp., and eight strains of Ureaplasma urealyticum (T-strain). A mixture of urea, Triton X-100, and dithioerythritol was used to solubilize sonically disrupted cells. PAGIF was performed in the range of pH 3 to 10. Protein patterns were carefully compared, demonstrating resolved and distinguishable species-specific protein bands. The eight serotypes of U. urealyticum (T-strain) gave identical protein patterns in the pH 3 to 10 range. The characteristic "fingerprints" of a species appeared to correlate with the biochemical nature and not the habitat in each case. Arginine-hydrolyzing species seemed to show more diverse focusing than those that ferment glucose, or prefer an acid environment. Characterization and identification of highly resolved species-specific proteins, ease of performance, and reproducibility of this method suggest that PAGIF might be employed as a taxonomic aid.     

Isoelectric focusing of boar spermatozoa.

The isoelectric points of washed spermatozoa from intact boars and from boars after removal of the seminal vesicles were determined using isoelectric focusing on natural pH gradients. Normal boar spermatozoa focused at a higher pH than spermatozoa from boars without seminal vesicles. The isoelectric point of the latter was increased to a value approaching normal by preincubation in normal seminal plasma. This indicates that seminal plasma alters the membrane surface charge of boar spermatozoa on ejaculation.     

Isoelectric focusing in Pevikon slabs

A mixture of Pevikon C-870 and Sephadex G-75 superfine is proposed for isoelectric focusing in granular beds. The beds are easy to prepare, and pH gradients are stable for 36 hr under the given conditions. Precise and clean fractionations are obtained with excellent recoveries of focused substances.     

Standardisation of Gymnema sylvestre R.Br. by high-performance thin-layer chromatography: an improved method

An improved high-performance thin-layer chromatographic (HPTLC) method for the standardisation of Gymnema sylvestre is reported. The method involves the initial hydrolysis of gymnemic acids, the active ingredients, to a common aglycone followed by the quantitative estimation of gymnemagenin. The present method rectifies an error found in an HPTLC method reported recently.     

Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing.

A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.     

Isoelectric focusing of oligopeptides: detection by specific stains.

By using ultrathin (350 micrometers) polyacrylamide gels, which at the end of the fractionation are pasted to filter paper and dried in an oven at 110 degrees C, and after isoelectric focusing it has been possible to detect oligopeptides in the di- to tetradecapeptide range, which could not be detected by protein staining techniques. This is achieved by developing a series of specific stains for the following amino acids: Arg, Tyr, His, Trp, Met and Cys. Except for Met and Cys, the detection limits appear to be in the order of 0.2--2 micrograms of free amino acid loaded in the gel. The Pauli reaction for His and Tyr and the Sakaguchi stain for Arg can be developed sequentially in the same gel, thus allowing the detection of four different amino acids since, under these conditions, also Trp reacts. Unfortunately, more general reactions, such as the permanganate, the 'Lowry' and the ninhydrin stains, cannot be utilized since the carrier ampholytes react very strongly with all these reagents.     

Isoelectric focusing by free solution capillary electrophoresis.

A reproducible, quantitative isoelectric focusing method using capillary electrophoresis that exhibits high resolution and linearity over a wide pH gradient was developed. RNase T1 and RNase ba are two proteins that have isoelectric points (pI's) at the two extremes of a pH 3-10 gradient. Site-directed mutants of the former were separated from the wild-type form and pI's determined in the same experiment. The pI's of RNase T1 wild-type, its three mutants, and RNase ba were determined for the first time as 2.9, 3.1, 3.1, 3.3, and 9.0, respectively. The paper describes the protocol for isoelectric focusing by capillary electrophoresis, as well as presenting data describing the linearity, resolution, limits of mass loading, and reproducibility of the method.     

Isoelectric focusing of cells using zwitterionic buffers.

A simple method of isoelectric focusing of cells is described. The pH gradient, superimposed on a density gradient, is developed by generating opposing concentration gradients of two zwitterionic buffers. The method can be used as a cell separation technique or as a means of characterizing the cell type on the basis of the focusing pH. Focusing is rapid and thus the method is of special advantage in its application to cells.