Thin-layer Polyacrylamide Gel Research Articles

Electrotransfer of Immunoprobes through Thin-Layer Polyacrylamide Gels.

Hydrogels are important structural and operative components of microfluidic systems, finding diverse utility in biological sample preparation and interrogation. One inherent challenge for integrating hydrogels into microfluidic tools is thermodynamic molecular partitioning, which reduces the in-gel concentration of molecular solutes (e.g., biomolecular regents), as compared to the solute concentration in an applied solution. Consequently, biomolecular reagent access to in-gel scaffolded biological samples (e.g., encapsulated cells, microbial cultures, target analytes) is adversely impacted in hydrogels. Further, biomolecular reagents are typically introduced to the hydrogel via diffusion. This passive process requires long incubation periods compared to active biomolecular delivery techniques. Electrotransfer is an active technique used in Western blots and other gel-based immunoassays that overcomes limitations of size exclusion (increasing the total probe mass delivered into gel) and expedites probe delivery, even in millimeter-thick slab gels. While compatible with conventional slab gels, electrotransfer has not been adapted to thin gels (50-250 μm thick), which are of great interest as components of open microfluidic devices (vs enclosed microchannel-based devices). Mechanically delicate, thin gels are often mounted on rigid support substrates (glass, plastic) that are electrically insulating. Consequently, to adapt electrotransfer to thin-gel devices, we replace rigid insulating support substrates with novel, mechanically robust, yet electrically conductive nanoporous membranes. We describe grafting nanoporous membranes to thin-polyacrylamide-gel layers via silanization, characterize the electrical conductivity of silane-treated nanoporous membranes, and report the dependence of in-gel immunoprobe concentration on transfer duration for passive diffusion and active electrotransfer. Alternative microdevice component layers─including the mechanically robust, electrically conductive nanoporous membranes reported here─provide new functionality for integration into an increasing array of open microfluidic systems.

In Situ Single‐Cell Western Blot on Adherent Cell Culture

AbstractIntegrating 2D culture of adherent mammalian cells with single‐cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single‐cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin‐functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one‐step copolymerization process that creates protein‐decorated microwells. After single‐cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell‐based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab‐gel western blot, while revealing cell‐to‐cell heterogeneity in stress responses.

In Situ Single-Cell Western Blot on Adherent Cell Culture.

Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one-step copolymerization process that creates protein-decorated microwells. After single-cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell-based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.

Transglutaminase-Induced Chemical and Rheological Properties of Cheese

Ca2+-independent transglutaminase isolated from Streptoverticillium mobaraense was used to obtain a novel cheese we called “crosslinked cheese.” We characterized both the cheese obtained by adding transglutaminase and milk clotting enzyme at the same time, and the one produced by adding transglutaminase, following cutting of the coagulum. In both cases, water content and cheese yields were higher, whereas protein content significantly increased from 33.8% to 39.5% only in the second type of crosslinked cheese. Isoelectric focusing of the protein extracted from the prepared cheese samples on a thin layer of polyacrylamide gel indicated that mainly β-caseins were involved in the transglutaminase-catalyzed crosslinks occurring in the curd. Few αs1-casein isoforms were entrapped in the curd matrix only when transglutaminase was added at the same time as the coagulant enzyme. Soluble nitrogen determination and HPLC peptide analysis indicated a markedly reduced proteolysis of crosslinked cheese during ripening, probably responsible for the observed enhancement of their hardness in comparison with the control cheese, obtained in the absence of transglutaminase.

Antibodies in oligoclonal immunoglobulins in CSF from patients with acute cerebrovascular disease.

Thin-layer polyacrylamide gel isoelectric focusing (PAG IEF) of CSF and serum, and subsequent immunofixation with viral and structural brain components followed by autoradiography revealed in eight out of nine selected patients with oligoclonal CSF IgG and cerebrovascular disease local synthesis within the CNS of antibodies against one or more of the viruses tested: six patients against measles, five against herpes simplex virus type 1, and two against varicella virus. This finding may reflect a polyclonal B cell activation secondary to brain damage and elaboration of certain structural brain components. None of the patients had local synthesis of antibodies against the other viruses tested (mumps, rubella and cytomegalovirus), or against structural brain components (crude saline, lipid-proteolipid, myelin basic protein extracts from human brain and purified bovine myelin basic protein).

Zymogram patterns of α-amylase isozymes inEthiopian tetraploid wheat landraces: insight into their evolutionaryhistory and evidence for gene flow

The variation for α-amylaseisozymes was analyzed in 71 tetraploid wheat (AABB genome)landraces from Ethiopia, including accessions of Triticumdicoccon Shrank, T.turgidum L., T.durum Desf., T.pyramidale Percival and T.aethiopicum Jakubz., by thin-layerpolyacrylamide gel isoelectric focusing. Four zymogram phenotypeswere obtained from all the materials studied. Except inT. dicoccon, however, thestandard pattern of the durum wheat variety, LD222, was predominant.T. dicoccon showed twozymogram types that differed for the absence or consistently weakactivity of band 18(α-Amy-B1)of the malt type. Band 1(α-Amy-B3)of the malt type was fixed in T.dicoccon and present in only 10% of thefree-threshing (FT) types. Generally, the resultsindicated that the variation forα-amylase isozymes in cultivatedtetraploid wheats, including landraces from secondary centers, is lowpossibly due to the founder effect or as a result of selection. Thedata were useful for inferences about the evolutionary history ofEthiopian wheat landraces. It is speculated thatT. dicoccon was the firstwheat to arrive in the Ethiopian highlands ca.5000 years ago. However, it is not known whether the present day FTEthiopian tetraploid wheat landraces are direct descendants ofT. dicoccon, or whetherthey were introduced independently. The clear differences inα-amylase zymogram patterns favor the latterhypothesis. However, the presence of band1 in some of the FT types indicated the occurrence of geneflow between the FT types and T.dicoccon, which may also explain thequantitative nature of spike threshability in these landraces.Possible implications for durum wheat breeding arediscussed.

Population Genetics of Two Tetranucleotide Repeats (UGB and D3S1349): A Preliminary Study in Galicia, Spain

Abstract The polymorphism of two tetranucleotide repeats, UGB and D3S1349 in the population of Galicia, Spain was carried out. Up to 7 and 8 alleles respectively were found, which determine values of H = 0.820 and H = 0.808 for these two STRs. A non-isotopic technique using thin layer polyacrylamide gel electrophoresis followed by silver staining, for the screening of both systems, is presented and discussed. Formal genetic studies from 51 meioses are concordant with an autosomal co-dominant way of inheritance of these systems.

Characterization of adult Dicrocoelium dendriticum by isoelectric focusing.

Water soluble extracts of 3131 adult specimens of Dicrocoelium dendriticum from cattle, sheep and goats, mainly from León province, were analysed by isoelectric focusing in thin-layer polyacrylamide gels. Activity of the following enzymes was studied: lactate dehydrogenase (LDH, EC 1.1.1.27), glucose phosphate isomerase (GPI, EC 5.3.1.9), phosphoglucomutase (PGM, EC 2.7.5.1), acid phosphatase (AcP, EC 3.1.3.2), alpha-glycerophosphate dehydrogenase (alpha-GPDH, EC 1.1.1.8), hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.30) and malate dehydrogenase (MDH, 1.1.1.37). Five distinct enzyme types were recognized for LDH (pH range 6.30-7.13), GPI (pH 6.13-6.80) and PGM (pH 6.20-6.60) whereas AcP showed three different patterns (pH 5.70-5.92). Weak and diffuse activity was detected for MDH (pH 4.8-6.2) and no activity was observed for alpha-GPDH and HBDH. In general, little phenotypic variation was observed between worms recovered from a single host, between those from hosts of the same species and between those from hosts of different species, although some enzyme types were found in some animals but not others. Nevertheless, it must be taken into account that most parasites came from sheep and also from a relatively small area in north-west Spain.

Using Isozyme Polymorphisms for Identifying and Assessing Genetic Variation in Cultivated Pawpaw [Asimina triloba (L.) Dunal

The utility of isozyme phenotypes for identifying and determining genetic variation in pawpaw cultivars was studied using isoelectric focusing in thin-layer polyacrylamide gels. Based on a sample of 32 clones (cultivars and advanced selections) and 23 enzyme systems, 7 enzymes were found to be polymorphic, involving 9 polymorphic loci [acid phosphatase (ACP), dihydrolipoamide dehydrogenase (DDH), malic enzyme (ME), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), peroxidase (PRX), and shikimate dehydrogenase (SKD)]. Altogether these 9 loci and 32 clones yielded 28 multi-locus isozymic phenotypes useful for cultivar identification; 24 of the 32 clones were uniquely identified. The allozyme variation in these clones has the average of other long-lived woody perennials of widespread geographic range in temperate regions with insect-pollinated outcrossing breeding systems, secondary asexual reproduction, and animal-dispersed seed. Genetic differentiation among these pawpaw clones, measured by Nei's distance, D, was substantial: 496 pairwise comparisons of genetic distance among the 32 clones indicated that they differed on average of D = 0.068 ± 0.04 and ranged from 0 to 0.188. Cluster analysis (UPGMA) produced a most likely division of the 32 clones into 7 groups; however, these groups did not conform to known pedigree relations. Additional polymorphic enzymes are needed for accurate allozyme-based genetic discrimination.

Low frequency of PIM3 gene in patients with monoclonal gammopathies.

The distribution of PI (protease inhibitor) phenotypes and PI M subtypes was studied in 200 patients with monoclonal gammopathies and 320 healthy blood donors by isoelectric focusing in thin-layer polyacrylamide gels, pH range 4-5. The distribution of PI phenotypes and gene frequencies in the patients differed significantly from the corresponding values found in the blood donors, as shown by the chi 2 test (chi 2 = 16.5, p = 0.02 and chi 2 = 17.6, p = 0.0014, respectively). A lower frequency of the M3 variant was observed in patients, both in homozygous (PI M3) and heterozygous forms (PI M1M3 and PI M2M3). No significant difference between PIz allele frequencies in patients and healthy controls was found.

Alpha-2-HS-glycoprotein (AHSG) polymorphism in semen and saliva.

Polymorphism of alpha-2-HS-glycoprotein (AHSG) was demonstrated in human semen and whole saliva samples by thin-layer polyacrylamide gel isoelectric focusing (IEF) and immunoblotting. Although the seminal AHSG IEF patterns were found to differ from those of plasma AHSG from the corresponding donors, incorporation of Nonidet P-40 into the IEF gel (pH 4.2-4.9) enabled us to phenotype seminal AHSG correctly. Salivary AHSG, however, exhibited IEF patterns similar to those of the corresponding plasma AHSG. By treating the samples with neuraminidase, it was possible to determine the AHSG types using 2-5 microL semen and 50-100 microL whole saliva samples. The AHSG types determined separately in 47 sets of semen, whole saliva, urine and plasma samples from the same donors correlated perfectly with each other. AHSG typing could, therefore, provide an additional discriminant characteristic in the forensic examination of semen and saliva samples.

Application of Isoelectric Focusing in Thin-Layer Polyacrylamide Gels to the Study of Opine Dehydrogenases in Marine Invertebrates

Application of Isoelectric Focusing in Thin-Layer Polyacrylamide Gels to the Study of Opine Dehydrogenases in Marine Invertebrates

Compound Heterozygous Mutations Affecting Both Hepatic and Erythrocyte Isozymes of Pyruvate Kinase

Novel erythrocyte pyruvate kinase gene defects were found in a patient without a family history of consanguinity. The polymerase chain reaction products of the R-type pyruvate kinase cDNA from the propositus contained two point mutations of Ser80 (TCC) → Pro (CCC) and Arg490 (CGG) → Trp (TGG). Allele-specific polymerase chain reaction of the genomic DNA revealed that this patient was a compound heterozygote. The mobilities of the patient′s L- and R-type pyruvate kinase by thin-layer polyacrylamide gel electrophoresis were abnormal. The results are consistent with the fact that these mutations are within exons common to the hepatic and erythrocyte isozymes.

The isolation and characterisation of jacalin [ Artocarpus heterophyllus (jackfruit) lectin] based on its charge properties

Jackfruit extracts contain a protein termed jacalin which possesses diverse biological properties. A detailed analysis of its charge properties has been lacking. The present investigation was initiated to study isoelectric properties of jacalin in detail and to isolate a single isoform of jacalin. Jacalin was isolated from jackfruit extracts by affinity chromatography on immunoglobulin-A immobilised to Sepharose 4B. Various techniques such as ion-exchange chromatography, isoelectric focusing (IEF) on polyacrylamide gels and preparative liquid IEF with the Rotofor cell were used. When analysed by IEF on thin layer polyacrylamide gels, jacalin was resolved into 35 bands over a pH range of 5.0–8.5. Upon SDS-PAGE in the second dimension all these charge species gave rise to only two-bands at 12 and 15.4 kDa. The lectin was mostly eluted with 50 and 100 mM sodium chloride when jackfruit extracts were fractionated on an anion-exchange column of DEAE-cellulose. In a single 6 hour run by preparative IEF with the Rotofor cell in the pH range of 3–9.5, it has been possible to isolate pure jacalin fractions containing fewer number of charged isomers. A single jacalin isoform was isolated by subjecting a Rotofor fraction containing fewer charged species to preparative IEF on thin layer polyacrylamide gel and eluting the band of interest from the gel. The isolated jacalin isoform was biologically active as it agglutinated erythrocytes. The study reveals the complexity of jacalin as it exists as multiple charge isomers over a broad pH range. By performing preparative IEF in solution as well as in thin layer polyacrylamide gels, it was possible to isolate a single jacalin isoform with the retention of biological activity.

The composition and properties of date proteins

Proteins from various date cultivars were isolated by extraction with phosphate-buffered saline (PBS) and were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Dates contained a number of proteins with molecular weights ranging from 12000 to 72000. Although there were minor protein bands, most date cultivars contained two prominent bands appearing at 30000 and 72000. Upon isoelectric focusing on thin-layer poly-acrylamide gels, date proteins were resolved into five or six bands in the pH range of 4.15–5.85. Sequential extraction of date pulps, first with water and later with PBS, showed that most date proteins were water-soluble albumins. When the kinetics of protein accumulation were studied, it was observed that early green dates contained very little protein. There was a rapid increase in the protein content and also in the number of protein components at a later stage in maturation. Dates from the countries of the Middle East such as Saudi Arabia, Oman and Iran were similar in their protein profiles as they contained similar complex mixtures of proteins in the molecular weight range of 12000–72000. A date variety from the USA had very little protein and also a simple protein profile with one major band appearing at 30000. Amino acid analysis revealed that dates. irrespective of their cultivars. contained all the essential amino acids. These proteins were rich in acidic amino acids and poor in sulphur-containing amino acids such as methionine and cysteine.

Determination of the Molecular Weight of Extracellular Ribonuclease Isoenzymes from Aspergillus Niger in Crude Extracts by Thin Layer Gel Filtration and Polyacrylamide Gel Electrophoresis

Abstract The extracellular ribonucleases from Asperqillus niger culture medium were fractionated according to their molecular weight by thin layer gel filtration through Sephadex G100 superfine and the enzyme activity was detected by a standard staining technique on a replica print paper. Another replica paper was laid onto the top of a polyacrylamide gel and the absorbed proteins were separated by electrophoresis. By comparing the electrophoretic pattern with that of a control not subjected to gel filtration, the molecular weight of each isoenzyme in the crude extract could be determined. Gel electrophoresis however, is only used to establish the correspondence between the original electrophoretic pattern of the isoenzymes in the crude preparation and that detected on the replica print paper taken after the thin layer gel filtration run. There was good agreement between the values obtained for the crude and purified enzymes.

The use of nuclear muprobe techniques to study the chemistry of lacustrine sediments and particles

The Oxford SPM has been used in two novel studies of lake chemistry: (a) The distribution of dissolved iron in sediment pore waters close to the sediment/water interface has been measured using the novel technique of diffusive equilibration in a thin film (DET). In this technique, which has a spatial resolution of < 1 mm, much less than that of competing techniques (1 cm), a thin layer of polyacrylamide gel is inserted into the sediment and after the rapid equilibration with the pore water, the gel is dried and fixed. The distribution of trace elements can then be measured using mubeam PIXE. Preliminary results have shown for the first time a subsurface maximum of Fe consistent with current theories of Fe dynamics. This paper presents some results obtained using the technique and discusses the limits on resolution and sensitivity, (b) Individual suspended lake particles (predominantly iron oxides and sulphides) have been analysed using point mubeam RBS and PIXE. Of particular interest in this study is the oxidation state of iron rich particles, so RBS with a 1 μm beam was used to determine the Fe: O stoichiometry of single particles. The particles were filtered from a depth of 14 m in Esthwaite Water in the English Lake District and handled in anoxic conditions until evacuation in the SPM sample chamber. Two distinct compositions of iron oxide were determined in clusters of about 5 μm diameter. Analysis by PIXE revealed that FeS was uniformly distributed in the particulate material and that it also contained elevated levels of Cu and Zn. This study was the first to demonstrate directly that discrete clusters of iron oxides are present in black particulate material which is commonly considered to comprise iron sulphides.

Genetic polymorphisms of α- and β-amylase isozymes in wild emmer wheat, Triticum dicoccoides, in Israel

α- and β-amylase isozyme diversity was studied electrophoretically by thin-layer polyacrylamide gel isoelectrofocusing in the tetraploid wild emmer wheat, Triticum dicoccoides, the progenitor of all cultivated wheats. We analyzed 225 plants from 23 populations encompassing the ecological spectrum of T. dicoccoides in Israel. The results were as follows: (a) Band and multilocus genotype polymorphisms abound and vary within and between the four amylase components: malt, green (α-amylases), and dry and germinating seeds (β-amylases). (b) The number of bands of malt, green, and dry and germinating seeds were 20, 6, 11 and 13, respectively, generating 40, 6, 51, and 51 patterns or multilocus genotypes (MGP), respectively. The MGPs vary drastically within and between populations, from monomorphic in some populations with a single pattern to highly polymorphic ones, (c) Mean H e values for malt, green, and germinating and dry seeds are 0.053, 0.055, 0.088, and 0.077, respectively; mean number of bands per individual was 11.8, 4.4, 7.6, and 4.0, respectively, (d) The percentages of 50 bands and 148 multilocus genotype patterns (MGP) (in parenthesis) were classified into widespread, sporadic, and localized: 84.4 (10.8), 8.9 (12.2), 6.7 (77.0), respectively. Notably, 89.2% of the patterns were not widespread, but sporadic and localized, (e) The mean value of genetic distances among populations (Nei's D) for the four amylase groups is D = 0.136, 0.175, 0.288 and 0.307, respectively, not displaying geographical correlates. (f) Most of the α- and β-amylase diversity is between populations (G st = 68-75%). (g) Significant environmental correlates occur between either bands or patterns and climatic diversity (water and primarily temperature factors). (h) Significant associations of multilocus amylase bands occur across Israel. Like-wise, significant gametic phase disequilibria, D, occur within populations and are positively correlated with climatic variables, primarily that of temperature, (i) Discriminant analyses correctly classified (95-100%) the 23 wild emmer populations into their ecogeographical region and soil type. (j) Autocorrelation analysis showed that there is no correlation between bands and geographic distance and excluded migration as a major factor of amylase differentiation.These results suggest that diversifying climatic and edaphic natural selection rather than stochastisity or migration is the major evolutionary force driving amylase differentiation at both the single and multilocus levels. Furthermore, wild emmer harbors high levels of α- and β-amylase diversity both as single bands and as multilocus adaptive genetic patterns. These are exploitable both as genetic markers for quantitative loci (QTLs) and as adaptive genetic resources to improve wheat germination and growth through classical breeding and/or biotechnology.

The zymogram method for detection of ribonucleases after isoelectric focusing: Analysis of multiple forms of human, bovine, and microbial enzymes

A zymogram method for detection of in situ ribonuclease (RNase) activity, combined with isoelectric focusing in a thin layer of polyacrylamide gel (IEF-PAGE), has been developed. After incubation with a dried agarose film containing substrate RNA, ethidium bromide, and an appropriate reaction buffer, which was placed tightly on the top of the focused gel, sharp and distinct dark bands corresponding to RNase isoenzymes on a fluorescent background appeared under uv light. Addition of urea to the IEF-PAGE gel at a final concentration of 4.8 m permitted optimal focusing of the RNases. This method had not only a high sensitivity of less than 0.1 ng purified RNase A, but also a high band resolution compared with the immunostaining method. It was also useful for analysis of purified enzymes, including bovine pancreatic RNases and two types of human urine RNase as mammalian enzymes, and RNases T 1 and T 2 as microbial enzymes, as well as for detection of RNases present in crude tissue extracts, resulting in more detailed elucidation of the multiplicity of these enzymes.

A new transferrin allele in Australian goats.

Subdivision of TF B into two variants, B1 (faster) and B2 (slower) in Australian goat breeds was accomplished by high voltage, thin layer polyacrylamide gel electrophoresis at pH 7.9. The genes controlling the caprine transferrins were shown to be autosomal codominant alleles, TFA, TFB1, TFB2 and TFC and in the various breeds of goats, the alleles were in Hardy-Weinberg equilibrium. TFA was the most common allele in the Australian and Texan Angora, Cashmere and Dairy breeds with gene frequencies ranging from 0.652 to 0.977. TFB1 and TFB2 occurred in all four breeds while TFC was only observed in very low frequencies in Australian Angora and Cashmere breeds.