Ultrathin Polyacrylamide Gels Research Articles

Isoelectric Focusing of Proteins in The pH Gradient As a Tool for Identification of Species Origin of Raw Meat.

IntroductionHealth, religious, and commercial aspects justify the need for meat species identification. The lack of officially approved methods prompts the undertaking of research on validation of isoelectric focusing of proteins (IEF) for official purposes.Material and MethodsSamples were prepared from pigs (Sus scrofa ferus domestica), cattle (Bos taurus), and poultry (Gallus gallus domesticus). Meat mixtures were made by blending 50%, 25%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.2% meat of other species. Samples were examined on ultrathin polyacrylamide gels with pH 3–9 gradient.ResultsThe results of the study confirmed the stable and reproducible pattern of meat protein bands. The detection limit of raw meat admixtures from pigs, cattle, and poultry mostly ranged from 2% down to 0.2% (0.2% for poultry). However, the IEF method can be used to detect the addition of pig meat to bovine meat in an amount higher than 3%. At the significant mixture level (i.e at least 5% addition of meat of another species) IEF proves itself with 100% specificity, sensitivity, and accuracy.ConclusionThe achieved detection limits provide a basis for recommending the IEF method for routine tests in laboratories detecting the species origin of meat.

High Sensitivity Top‐down Proteomics: Coomassie for In‐gel Proteoform Detection Rivals MS‐based Peptide Detection

Several thousands of gene and splice variants, as well as post‐translational modifications, deepen the immense complexity of the human proteome. As these protein species or proteoforms define biological mechanisms in health and disease their direct analysis is critical, necessitating top‐down assessments in which the complete amino acid sequence and all modifications remain intact. Two‐dimensional gel electrophoresis (2DE) coupled with downstream mass spectrometry (MS) remains the most thorough and practical approach to Top‐down Discovery Proteomics, with resolving power to routinely analyze complex mixtures of intact proteoforms [1]. Increasing the quality and quantity of 2DE‐derived data largely relies on developing, optimising, and rigorously characterising methods for in‐gel total protein detection, providing quantitative, sensitive detection over a wide dynamic range while remaining practical [2]. Modified colloidal Coomassie Brilliant Blue (cCBB) staining meets these criteria [3, 4]; cCBB provides (sub)femtomole detection sensitivity for SDS‐PAGE and 2DE‐resolved intact proteoforms using either near‐infrared fluorescence detection (nIRFD) or densitometry [5]. This thus rivals the detection sensitivity of peptides by MS in ‘bottom‐up’ (i.e. shotgun) approaches to proteomics, which appear to provide larger protein catalogues but at the expense of critical data concerning proteoforms. Our ongoing work addresses a number of experimental variables which impact detection sensitivity in standard 1 mm‐thick polyacrylamide gels, providing a more realistic baseline measure of cCBB detection sensitivity and enhancing the overall practicality of 2DE for comprehensive proteome analysis. Here we showcase our most recent findings, including preliminary findings of further improvements to cCBB detection sensitivity when using ultra‐thin polyacrylamide gels. The novel ultra‐thin method utilised has been developed from minimally modified commercial equipment for vertical mini‐format gel electrophoresis and is thus accessible, affordable, and simple to implement in laboratories equipped for standard SDSPAGE and 2DE. The ultra‐thin gels do not require backing supports for handling, and potentially are compatible with commercial immobilised pH gradient strips routinely used for first dimension isoelectric focussing, further adding to the value and practicality of the method. Ultra‐thin 2DE may thus enable further mining of 2DE‐resolved proteomes, including the application of high resolution gel‐based proteomics in situations of limited sample quantity (e.g. biopsies, and perhaps single cells).Support or Funding InformationNN and PSA received scholarship support from the WSU Molecular Medicine Research Group and the WSU School of Medicine, respectively. JRC notes the support of an anonymous private family foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gel for Simultaneous Chemical Imaging of Anionic and Cationic Solutes Using Diffusive Gradients in Thin Films

We report on a novel gel based on diffusive gradients in thin films (DGT) for the simultaneous measurement of cations and anions and its suitability for high resolution chemical imaging by using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). The new high resolution mixed binding gel (HR-MBG) is based on zirconium-hydroxide and suspended particulate reagent-iminodiacetate (SPR-IDA) as resin materials which are embedded in an ether-based urethane polymer hydrogel. The use of this polymer hydrogel material allows the production of ultrathin, highly stable and tear-proof resin gel layers with superior handling properties compared to existing ultrathin polyacrylamide gels. The gel was characterized regarding its uptake kinetics, the anion and cation capacities, and the effects of pH, ionic strength, and aging on the performance of the HR-MBG. Our results demonstrate the capability of this novel gel for concomitant sampling of anions and cations. The suitability of this new gel type for DGT chemical imaging at submm spatial resolution in soils using LA-ICPMS is shown. 2D images of P, As, Co, Cu, Mn, and Zn distributions around roots of Zea mays L. demonstrate the new opportunities offered by the HR-MBG for high-resolution mapping of solute dynamics in soil and sediment hotspots, such as the rhizosphere, by simultaneous observation of anionic and cationic solute species.

Distribution of α<sub>S1</sub>-casein "welsh" variant in some Slovak and Czech sheep breeds

Abstract. Occurrence of the αS1-casein D allele (formerly "welsh" variant) in sheep breeds Tsigai, Improved Valachian and Slovak Merino was investigated by means of isoelectric focusing in ultra thin polyacrylamide gel. This variant in herds of the above mentioned breeds was observed as follows: Improved Valachian 3.64%; Tsigai 5.57%; Slovak Merino 12.45%. In the last mentioned breed in a single animal αS1-casein was not found at all (0.34% of the total). There is no mention of existence of null allele of sheep αS1-casein in literature so far. In case of the Tsigai breed in one individual (0.24%) the "welsh" variant in homozygous form was observed.

Polymorphisms in blood proteins of Bos indicus and Bos taurus cattle breeds of Cameroon and Nigeria, and description of new albumin variants.

Polymorphisms in the five blood protein loci albumin (ALB), carbonic anhydrase (CA II), vitamin D binding protein (GC), haemoglobin (HBB), and transferrin (TF) were investigated in 520 individuals from 12 cattle populations (Bos indicus and Bos taurus) in Cameroon and Nigeria by isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels (PAG-IEF) and by linear gradient polyacrylamide gel electrophoresis (PAGE). While all loci in nine populations were polymorphic with up to six alleles at the ALB and TF loci: the Namchi population showed monomorphism at the CA II locus and Muturu at the ALB, CA II, and HBB loci. There was a clear distinction between Bos indicus and Bos taurus breeds at the ALB locus with ALBB predominating in indicine and ALBA predominating in taurtine breeds. CA IIS, GCA, and HBBA were the most commonly occurring alleles in all populations. Two variants not described before were demonstrated by PAG-IEF at the ALB locus and named ALBJ and ALBK. Mean effective number of alleles as measure of intrabreed diversity was higher in zebu populations (2.040-2.288) as compared to taurine breeds (1.349-1.836). Significant deviations from Hardy-Weinberg equilibrium occurred in some populations at the HBB and TF loci. More haplotypes of ALB/GC occurred in the zebu than taurine breeds. ALBAGCA predominated in the taurine populations and ALBBGCA in the indicine populations. Influence of zebu genes on the Namchi and N'Dama taurine breeds was detected at the ALB, CA II, HBB, and TF loci, and estimated at 61.5% and 5.7%, respectively. The high resolution of PAG-IEF in screening for polymorphisms within diversity studies was demonstrated.

Genetic polymorphism of goat kappa-casein (CSN3) in different breeds and characterization at DNA level.

Data on genetic differences at CSN3 in goat breeds including a DNA based typing method and the mutations responsible for variation on protein and DNA level are presented here. Isoelectric focusing (IEF) in ultrathin polyacrylamide gels with carrier ampholytes was used to demonstrate CSN3 polymorphism in milk samples of Italian (Orobica n=88; Ionica n=68) and German goat breeds (Bunte Deutsche Edelziege n=244; Weisse Deutsche Edelziege n=134; Toggenburger n=25; Thüringer Waldziege n=70). A further CSN3 band was found presenting a more cathodic migration than CSN A. After chymosin action, the genetic polymorphism was also observed in the para-kappa-casein fraction. The new allele CSN3(B) was spread mainly in Orobica (37%), Bunte Deutsche Edelziege (11%) and Ionica (10%). CSN3(B) occurred in low frequency (<3%) in Thüringer Waldziege and in Weisse Deutsche Edelziege, and could not be demonstrated in milk samples of Toggenburger. The populations were in Hardy-Weinberg equilibrium at CSN3. The codominant genetic control of CSN3(B) was confirmed by genetic studies. Discrimination of CSN3 alleles A and B was also obtained by DNA SSCP analysis. Sequencing of CSN3(B) revealed four transitions at position 247, 309, 471, and 591 compared with CSN3(A). From these transitions, the following amino acid substitutions are deduced: 44 Gln --> Arg, 65 Val --> Ile, 119 Val --> Ile, and 159 Ser --> Pro. Among the four mutations, only the 44 Gln --> Arg can be revealed by milk protein IEF analysis while at DNA level three further genetic variants should exist in addition to CSN3(A) and CSN3(B).

Genetic Polymorphism of Group-Specific Component/Vitamin-D-Binding Protein Subtypes in Six Populations from North-Eastern China

On six populations from North-Eastern China the frequencies of Gc subtypes were studied by isoelectric focusing on ultra-thin polyacrylamidegel followed by the improved sulfosalicylic acid precipitation. The following Gc subtype allele frequencies could be observed in the Han (Harbin), Ewenki, Tahur, Mongolian, Oroquen and Xibe populations: Gc*1F = 0.4246, 0.4941, 0.4479, 0.4077, 0.4606 and 0.4503; Gc*1S = 0.2587, 0.2936, 0.3151, 0.2822, 0.3370 and 0.3035; Gc*2 = 0.3065, 0.2064, 0.2266, 0.3006, 0.2022 and 0.2388. In addition to this 16 individuals of five populations showed rare Gc variant alleles (1A3, 1A8, 1A9, 1A14, 1C?).

Simple miniaturized gel system for DNA sequence analysis

A simple miniaturized gel system suitable for DNA sequencing is described. Small ultrathin polyacrylamide gels are cast, eight or more at a time, using standard microscope slides. Gels, ready to use, can be stored for approximately 2 weeks. Gels are run horizontally in a standard mini-agarose gel apparatus. Typical run times are 6-8 min. A novel sample loading system permits volumes of standard sequencing reactions as small as 0.1 microl to be analyzed. Sequencing ladders were visualized using 35S-labeled DNA by autoradiography and by colorimetric detection. Band resolution compares favorably with that of large gels. The methods introduced here serve as a step toward the miniaturization of DNA sequencing and are amenable to automated sample loading and detection.

An Improved High Resolution Single Method for Orosomucoid ORM1 and ORM2 Phenotyping

An improved high resolution single method for orosomucoid ORM1 and ORM2 phenotyping using isoelectric focusing (IEF) in wide-scales ultrathin layer polyacrylamide gels of pH range 4.2 to 4.9 is presented. The method is reliable, simple, and provides an alternative for the three currently required ones for typing this genetic system.

Effective blotting of ultrathin polyacrylamide gels anchored to a solid matrix.

Ultrathin polyacrylamide gels bound on glass plates or plastic sheets cannot be removed from their support without destruction. Therefore electrophoretic transfer methods are not applicable. We have developed a fast diffusion blotting procedure which is very simple and does not need any equipment like blotting chamber or power supply. Furthermore, no special buffer solutions are required. The method is universally applicable to ultrathin sodium dodecyl sulfate, native as well as isoelectric focusing polyacrylamide gels.

タマラオ(Bubalus (B.) mindorensis)ヘモグロビンの等電点電気泳動法による表現型とアジアスイギュウのそれとの比較

Hemoglobins (Hb) from seven captive tamaraws were examined and compared to that of water buffaloes (Bubalus bubalis): five river type and eleven swamp type; and two species of anoas: two lowland anoas (Bubalus (Anoa) depressicornis H. Smith, 1827) and eight mountain anoas (B. (A.) quarlesi Ouwens, 1910) using ultrathin polyacrylamide gel isoelectric focusing (PAGIF). The Hb phenotype of the tamaraws displayed a monomorphic single band, compared to two bands (major and minor bands) in water buffaloes and one to three allelic bands found in polymorphic anoa individuals. We designate this band HbTamaraw, having the highest isoelectric point (pI) focused at the same level as the minor band. Dissociation of globin chains by urea-PAGIF showed that HbTamaraw consists of a single type of α and β globins, whose α focused at the same pI level as the α minor band of water buffalo, as it is also true between the single α band of the anoa and theα major band of water buffalo focused at a lower pI. The β band of the tamaraw shared the same pI level to the β hands of both the water buffaloes and lowland anoas. The HbTamaraw differs by having a single α component from that of the water buffaloes, and an a band with a higher pI level from that of the anoas.

Epidemiology of Pseudomonas cepacia colonization among patients with cystic fibrosis.

Colonization with Pseudmonas cepacia in patients with cystic fibrosis (CF) has been associated with increased morbidity and early death, compared with colonization by P. aeruginosa. The mode of acquisition of P. cepacia is not fully understood, although person-to-person spread appears likely. Recent epidemiologic studies support the importance of social contact in the spread of P. cepacia among patients with CF. This study was undertaken to investigate the epidemiology of P. cepacia colonization among patients with CF attending the CF clinic at our center. Isolates of P. cepacia were collected from patients at two CF treatment centers, including ours. Additional isolates were collected from patients without CF in the hospital ICU, from other teaching hospitals, and from the environment. Profiles of enzymes were obtained by ultrathin polyacrylamide gel electrophoresis of P. cepacia extracts. A predominant electromorphic type (ET) was found among P. cepacia isolates from patients at both centers, suggesting a common source or person-to-person transmission. The majority of hospital isolates fell into a single, different ET. Surveillance swabs of respiratory equipment at our CF clinic did not grow P. cepacia. Attendance of patients at CF summer camp correlated strongly with P. cepacia colonization (P < 0.0001).

Isoelectric focusing of humic substances on ultrathin polyacrylamide gels: evidence of fingerprint performance

Differentiated interactions between humic substances and isoelectric focusing-carrier ampholytes were demonstrated by comparing specific absorption ratios from the supernatant after precipitation with 2 M HCl and with acid ampholytes (pH 2–4) containing both carboxylic and sulphate groups. An increasingly stronger interaction was observed when comparing soil humic substances to those of synthetic origin or those extracted from young reclaimed soil material. Electrofocusing of the humic substances under study revealed a fingerprint-like quality; the band patterns observed were consistent with the concept of three main fractions.

Multilocus enzyme electrophoresis using ultrathin polyacrylamide gels

Multilocus enzyme electrophoresis (MEE) using starch gels has been widely used to study bacterial genetics and for epidemiological typing. We describe a technique using ultrathin polyacrylamide gel eletrophoresis (PAGE) which gives improved resolution and reproducibility, decreasing the number of gels which have to be run. We also describe a technique whereby several enzymes may be developed from a single running gel, resulting in significant time savings.

Distribution of ITI phenotypes in the Iberian Peninsula: Negative correlation between ITI*3 frequencies and latitude in human populations.

The molecular phenotypes of ITI were analysed in samples from seven populations of the Iberian Peninsula (Galicia, Central Portugal, Castilla-Leon, Castilla-La Mancha, Extremadura, Western Andalusia, and Eastern Andalusia) by means of IEF in ultrathin layer polyacrylamide gels followed by immunofixation-silver staining. The observed ITI*3 frequencies exhibit the highest values thus far reported in European populations. Regression analysis between ITI*3 frequencies and latitude reveals a negative correlation (r = -0.8798, P < 0.001). © 1994 Wiley-Liss, Inc.

An improved method for typing of plasma alpha 1-protease inhibitor (PI1) variants in sheep.

Sheep plasma alpha 1-protease inhibitor (PI1) variants were typed by analysing neuraminidase treated samples by isoelectric focusing in ultrathin polyacrylamide gels followed by staining for trypsin inhibition. The 10 different PI1 phenotypes observed were shown to be controlled by four codominant alleles. The probability of sire exclusion provided by PI1 polymorphism was about 0.45 in both of the studied milk sheep breeds (Latxa and Karrantzar) of the Basque Country.

Isozyme analysis of Kenyan phlebotomine sandflies (Diptera: Ppsychodidae) by isoelectric focusing (IEF) on pharmacia phast system™

The isoenzyme profiles of nine sandfly species ( Phlebotomus duboscqi, P. elegonensi, P. martini, P. pedifer, Sergentomiya antennatus, S. bedfordi, S. garnhami, S. graingeri and S. schwetzi) from Kenya collected from the field and reared in the Medical Vectors Research Programme of the ICIPE were determined by isoelectric focusing (IEF) on mini ultra-thin polyacrylamide gels supplied by Pharmacia. Three of the nine isoenzymes screened (MDH, G-6-PD, PGM, ME, ICD, GAPDH, 6-PDG, HK and GPI), namely GPI, PGM and ME, gave banding patterns of taxonomic value. Cluster analysis of the simple matching coefficients gave two distinct groupings corresponding to previously described Sergentomyia and Phlebotomus respectively. However, S. garnhami, previously grouped in the genus Sergentomyia, was found to be associated with the genus Phlebotomus.

Flow cytometry and sorting of amphibian bladder endocytic vesicles containing ADH-sensitive water channels.

The water permeability of ADH target epithelial cells is believed to be regulated by a cycle of exo-endocytosis of vesicles containing functional water channels. These vesicles were selectively labeled in intact frog urinary bladders with an impermeant fluorescent marker, 6-carboxyfluorescein. Vesicle suspensions containing the labeled endosomes were obtained by homogenization and differential centrifugation of bladder epithelial cells. The osmotic permeability of the endocytic vesicles was measured, using a stopped-flow fluorescence technique, in the absence or in the presence of HgCl2. This permeability was found very high (500 microns/sec) and inhibited by 1 mM HgCl2 (90%), thus confirming the presence of water channels. The labeled endosomes were then separated from the other membrane vesicles by flow cytometry and sorting. Their protein content was analyzed by electrophoresis on ultrathin polyacrylamide gels. Two double bands were found at 71 and 55 kDa as well as a small band at 43 kDa. They respectively correspond to 31, 38 and 10% of the total amount of silver-stained proteins present in the sorted endosomes, while they only represent 2, 4, and less than 1% of the proteins contained in the vesicle suspension, before sorting. These highly enriched proteins (or at least one of them) are likely to be involved in the mechanism of water transport. Associated to their partial purification by differential centrifugation, the sorting of the endosomes by flow cytometry seems a good way to further characterize the water channel.

Genetic polymorphism of human factor H (HF, beta 1H globulin) in Chinese Han population in northeast China.

The distribution of human factor H of serum phenotypes were studied using ultrathin polyacrylamide gel isoelectric focusing (PAGIEF) and subsequent immunoblotting techniques in 203 Chinese of Han population in Liaoning Province of northeast China. The gene frequencies of HF*A and HF*B were 0.4828 and 0.5172, respectively. All the observed numbers of the phenotypes were in agreement with the expected numbers under the Hardy-Weinberg equilibrium. The gene frequencies among Chinese, Japanese, and Caucasian populations were compared.

Typing of human red cell and serum mixtures in three electrophoretic systems

The isoenzymes erythrocyte acid phosphatase and phosphoglucomutase were typed in mixed red cell samples which had been derived from two individuals; the protein group specific component was typed in mixed serum samples. Typing was performed by isoelectric focusing on ultrathin polyacrylamide gels. Depending upon the mixture, from 2 to 20% (but typically 5-10%) by volume of a second blood or serum needed to be present in a mixture before it could be detected. In the majority of cases when there was significant mixing, samples were readily identified as a mixture when the results consisted of unusual band patterns or unusual band intensities. There would be a few instances when blood or serum could not be identified as a mixture when masking effects occurred or when the mixture produced a combined, apparently normal, pattern.