Homogeneous Polyacrylamide Gel Research Articles

Adaptation of Stadier's apparatus for electrophoresis of main milk proteins

The object of research is a Stadier-type apparatus for analytical electrophoresis of proteins. In the milk proteins research, in addition, there is a need to carry out serial express analyzes of their various groups, as well as the isolation of individual homogeneous fractions. The dimensions of working chambers for analytical, express, and micro preparative electrophoresis of caseins and milk whey proteins were proposed to solve this task. For each type of electrophoresis, different chambers and formers are used without changing the design of the apparatus. The apparatus is suitable for electrophoretic systems used for the analysis of milk proteins. Analysis of casein in the anodic system of a homogeneous polyacrylamide gel in the presence of urea allows identification of the main fractions: αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, αS2-CN-13P, β-CN-5P, ϰ-CN-1P and three β-casein fragments f(29-209), f(106-209) and f(108-209). Express electrophoresis in the presence of urea reveals four fractions of caseins: αS1-CN, αS2-CN, β-CN, and ϰ-CN. The analysis of whey proteins in the Davis native disc electrophoresis system allows identification of β-Lg A, β-Lg B, α-La, BSA fractions, and a group of immunoglobulin fractions. The express electrophoregram differs by a common band A and B variants of β-Lg. Due to an adequate selection of electrophoretic systems, it is possible to identify semi-quantitatively all the main fractions of milk proteins under analytical or express mode. The adapted apparatus also makes it possible to conduct micro preparative electrophoresis and obtain the main fractions of milk proteins. In this case, the yield of electrophoretically pure proteins is: β-CN-5P (23±5 %), β-Lg (A+B) (27±6 %), α-La (11±3 %), and purified groups of αS1-CN-8P+αS1-CN-9P (25±6 %), αS2-CN-(10-13P) (6±1.5 %) and ϰ-CN-1P (7±2 %). The apparatus could be used at enterprises producing dairy protein products

Electrophoretic system for express analysis of whey protein fractions

Fractional specificity of biological action and ability to form bioactive peptides, which have a positive effect on different physiological systems of the body in the processes of proteolysis and digestion, are characteristic for whey proteins. Prospects for the production and application of whey protein fractions are related to the necessity of their composition control. The comparative analysis of the electrophoretic systems previously used for the milk protein analysis was carried out for the creation of an express analysis method of whey protein fractions. These are the anode disc electrophoresis system in the presence of sodium dodecyl sulfate, the Davis disc electrophoresis system for acidic proteins in native conditions, the system in a homogeneous polyacrylamide gel with urea. The Davis disc electrophoresis system for acidic proteins was chosen as the basis. For the adaptation of this system to the requirements of express analysis, the stacking polyacrylamide gel was removed from its composition and the concentration of the separating gel was reduced. The difference in the composition of electrode buffer and gel buffer ions was used to provide the high separation efficiency of protein fractions. This allows saving the effect of protein concentration in the whey sample on the first stages of electrophoresis. The position of the basic whey protein fractions on electrophoregrams was established with the help of homogeneous marker proteins (β-lactoglobulin and whey albumin). Аn accessible electrophoresis system in the slabs of a homogeneous polyacrylamide gel for serial express analysis of the fractional composition of whey proteins has been proposed as a result of researches. The system allows reliable identification of four protein fractions (α-LA, β-LG, BSA and IG). Close average values and standard deviations of the content of these fractions in 15 whey samples of one milk batch, obtained by the densitometry of three electrophoregrams: β-LG (37.3±4.2, 36.5±2.8; 38.3±2.7), α-LA (15.8±1.5, 15.8±1.3, 16.4±1.1), BSA (8.2±1.1, 8.0±1.0, 9.4±1.1), IG (17.6±1.9, 17.4±1.5, 16.8±1.5) testify about good reproducibility of the method. The proposed method may be useful for the express identification of the basic whey protein fractions, which are precursors of biologically active peptides.

ELECTROPHORETIC SYSTEMS FOR PREPARATIVE FRACTIONATION OF PROTEIN PRECURSORS OF BIOACTIVE PEPTIDES FROM COW’S MILK

The article considers the possibility of obtaining purified fractions-precursors of bioactive peptides from milk proteins by the method of preparative electrophoresis. To choose an electrophoretic system, a comparative study has been carried out of four methods of electrophoresis in polyacrylamide gel that are used to analyse milk proteins (disc-electrophoresis without disaggregating agents, and disc-electrophoresis in the presence of sodium dodecylsulfate in homogeneous and gradient gel, and electrophoresis in homogeneous gel with urea). Electrophoresis of the total milk protein has shown that none of these systems allows separating effectively all protein precursors of bioactive peptides. The next stage was obtaining two main groups of milk proteins – caseins and serum proteins for electrophoretic fractionation. With the help of analytical electrophoresis, it has been established that each of the obtained groups had a typical proteins composition. Then, the proteins groups obtained were fractionated by preparative electrophoresis using the four electrophoretic systems listed above. In this case, the casein proteins that differ in the primary structure (αS1-, αS2-, β-, and ϰ-caseins) can be effectively separated by preparative electrophoresis on the basis of a homogeneous gel system in the presence of urea. The composition of this electrophoretic system was simplified. Unlike the analytical variant of a homogeneous polyacrylamide gel system, the toxic 2-mercaptoethanol was excluded, and the urea concentration was reduced. For the fractionation of serum proteins, a disc-electrophoresis without disaggregating agents can be used as a basis. It allows obtaining the main precursors of bioactive peptides from milk serum proteins: β-lactoglobulin, α-lactalbumin, serum albumin, and immunoglobulins. The protein precursors obtained by preparative electrophoresis were used to develop the biotechnology of obtaining bioactive phosphopeptides and inhibitors of the angiotensin-converting enzyme.

Identification of protein fractions of milk cows casein complex

To date, dozens of biologically active peptides formed during proteolysis of casein fractions have been discovered. The use of these peptides is closely related to the necessity of their rapid identification. The aim of this work was the development of an electrophoresis system for rapid identification of individual fractions in serial studies and the separation of the milk casein complex. Considering the abnormal nature of the interaction of caseins with the sodium dodecyl sulfate and similar values of their molecular masses, the anode electrophoresis system in a homogeneous polyacrylamide gel was taken as a basis. Caseins, in this system, are separated according to their charge and located on the electrophoregram in accordance with the modern classification. Urea was used as a disaggregating agent in gel. It was shown that the use of Studier type apparatus for electrophoresis with changeable dimensions of electrophoretic chamber significantly reduces (to 45 min) the time for identification of casein fractions. This method may be useful for rapid identification of casein fractions, as well as for rapid analysis of natural milk and milk products.

Structure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuni

Mass spectrometry investigations of partially purified Campylobacter jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan with a mass of 1406 Da and composed of one hexose, five N-acetylhexosamines and a species of mass 228 Da, consistent with a trideoxydiacetamidohexose. By means of soybean lectin affinity chromatography, a mixture of glycoproteins was obtained from a glycine extract, and two-dimensional gel proteomics analysis led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins. Glycopeptides were prepared from the glycoprotein mixture by Pronase digestion and gel filtration. The structure of the glycan was determined by using nano-NMR techniques to be GalNAc-alpha1,4-GalNAc-alpha1,4-[Glcbeta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-beta1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing further evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the biosynthesis of bacillosamine.

Novel cross-linked homogeneous polyacrylamide gels with improved separation properties: investigation of the cross-linker functionality.

Polyacrylamide (PAAm) gels were synthesized using cross-linkers with their potential functionality (twice the number of double bonds of a cross-linker) varying from six to sixteen. Improved electrophoretic separation and highly desirable porosity and sieving properties were observed for most of the PAAm gels containing novel cross-linkers. An increase in the potential functionality of cross-linkers used in PAAm gels was an important factor, influencing the pore size and pore size distribution of the network.

Microelectrophoresis for the separation of DNA fragments.

A methodology has been developed which significantly reduces the linear dimension necessary for the electrophoretic separation of DNA fragments and oligonucleotides. DNA fragments are rapidly separated into compact, resolvable microscopic banding patterns which can be detected using a high-resolution electronic imaging system. Separations can be carried out in either capillary tube or thin-layer (slab) microgel formats of one centimeter or less in length. The complete separation of all eleven fragments (1353 to 72 base pairs) of the pi X174 DNA/HaeIII restriction ladder was achieved in a total running distance of less than 2 mm and in less than 2 min. The observed band widths for the larger fragments (1353-603 bp) ranged from 18 to 25 microns, with the intermediate and smaller fragments (310 to 72 bp) ranging from 30 microns to 60 microns. The ethidium bromide-stained microgels were analyzed using an epifluorescent microscope combined with an intensified charged coupled device imaging system. In other experiments, single-base resolution of fluoresceinated oligonucleotides in the 20-30 nucleotide range was demonstrated. DNA sequencing may be possible with further optimization. This new methodology departs from the conventional gel formulations and electrophoretic procedures used for the separation DNA fragments. High voltage gradients and the use of highly concentrated and crosslinked homogeneous polyacrylamide gels effects the rapid separation of DNA fragments in very short distances. Analysis of the microgels with proteins of known size (Stokes radius) indicates that separations are occurring in gels with pore sizes close to the diameter of double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of Haemophilus influenzae type b subtypes by numerical analysis.

Outer membrane proteins (OMPs) were prepared from 20 previously established subtypes of Haemophilus influenzae type b. Proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (11% acrylamide). Each lane in the gels contained two internal molecular weight standards. One central lane contained a range of molecular weight standards, which were used to calculate a molecular weight curve for each gel. Migration distances of the OMPs were determined with a soft laser-scanning densitometer, and the distances were normalized by using the mean migration distances of the internal standards. The protein patterns of all subtypes were compared by a recently described method (B.D. Plikaytis, G.M. Carlone, and B.B. Plikaytis, J. Gen. Microbiol. 132:2653-2660, 1986). All subtypes could be differentiated by this method. The ability to store and compare numerous OMP patterns from different isolates of H. influenzae type b, separated with a single homogeneous polyacrylamide gel, will facilitate the continued development of a subtyping system based on these proteins.

Activity and electrophoretic multiplicity of molecular forms of superoxide dismutase in human blood cells

The level of superoxide dismutase (SOD) activity calculated relative to protein in the various human blood cells falls in the following order: platelet > erythrocyte > lymphocyte > granulocyte. During electrophoresis in homogeneous polyacrylamide gel of homogenates of granulocytes, lymphocytes, and platelets three zones of SOD activity were identified. Two fractions of the enzyme, disappearing after treatment with cyanide, were found in lysates of erythrocytes after removal of the hemoglobin. Of the two SOD fractions of platelets, lymphocytes, and granulocytes migrating rapidly toward the anode, the first corresponds in its ability to be inhibited by cyanide or organic solvents to the cytosol isozyme, the second to the mitochondrial isozyme. The third cathode fraction was not identified. The functional role of SOD in specialized blood cells and the cause of heterogeneity of the enzyme are discussed.