A Simultaneous Visualization of the Antioxidant Enzymes Glutathione Peroxidase and Catalase on Polyacrylamide Gels

A simple and sensitive method for the simultaneous visualization of glutathione peroxidase and catalase on polyacrylamide gels is described. The procedure included: (I) running samples on a 7. 5% polyacryla-mide gel, (2) soaking the gel in a certain concentration of reduced glutathione (0.25-2.0 mM). (3) soaking the gel in GSH plus H_zO_z or cumene hydroperoxide, (4) finally staining with a 1% ferric chloride I% potassium ferricyanide solution. The best concentration of glutathione for simultaneous visualization of glutathione peroxidase and catalase was 0.25rnM; I.5mM glutathione was the best concentration for visualization of glutathione peroxidase alone. The method is sensitive enough to detect catalase and glutathione peroxidase in mouse liver homogenates and also it is specific for glutathione peroxidase since other peroxidases such as lactoperoxidase, horseradish peroxidase and glutathione S-transferase cannot be visualized. Using this method, it was found that unlike catalase. glutathione peroxidase is heat resistant (68°C. 1min), but sensitive to 10mM sodium iodoacetate.     

A Simultaneous Visualization of the Antioxidant Enzymes Glutathione Peroxidase and Catalase on Polyacrylamide Gels

A simple and sensitive method for the simultaneous visualization of glutathione peroxidase and catalase on polyacrylamide gels is described. The procedure included: (I) running samples on a 7. 5% polyacryla-mide gel, (2) soaking the gel in a certain concentration of reduced glutathione (0.25–2.0 mM). (3) soaking the gel in GSH plus H_zO_z or cumene hydroperoxide, (4) finally staining with a 1% ferric chloride I% potassium ferricyanide solution. The best concentration of glutathione for simultaneous visualization of glutathione peroxidase and catalase was 0.25rnM; I.5mM glutathione was the best concentration for visualization of glutathione peroxidase alone. The method is sensitive enough to detect catalase and glutathione peroxidase in mouse liver homogenates and also it is specific for glutathione peroxidase since other peroxidases such as lactoperoxidase, horseradish peroxidase and glutathione S-transferase cannot be visualized. Using this method, it was found that unlike catalase. glutathione peroxidase is heat resistant (68°C. 1min), but sensitive to 10mM sodium iodoacetate.     

Hlad Immobilization in Polyacrylamide Gels

The immobilization of horse liver alcohol dehydrogenase in a cross-linked acrylamide-N,N¹-methylenebisacrylamide copolymer gel is described. The influence of monomer concentration, the degree of cross-linking and the polymerization technique on enzyme entrapment is studied. A bead-polymerization process produced the most useful and stable immobilized enzyme preparations.     

Hlad Immobilization in Polyacrylamide Gels

The immobilization of horse liver alcohol dehydrogenase in a cross-linked acrylamide-N,N¹-methylenebisacrylamide copolymer gel is described. The influence of monomer concentration, the degree of cross-linking and the polymerization technique on enzyme entrapment is studied. A bead-polymerization process produced the most useful and stable immobilized enzyme preparations.     

聚丙烯酰胺凝胶固定核酸方法及其应用

固相核酸已被广泛用于DNA/cDNA微阵列、固相PCR及其它核酸与生物分子检测的传感技术中.和硬质玻璃载片相比,三维聚丙烯酰胺凝胶作为固定核酸的载体具有结合核酸容量高、利于反应的类似液相环境和较少的空间效应等优点.综述了丙烯酰胺凝胶作为固定核酸载体的发展历史.着重介绍了丙烯酰胺修饰核酸直接聚合固定的方法以及在DNA芯片、焦测序、固相PCR(克隆)、及全基因组测序等核酸分析中的应用.     

In Situ Activity of Enzymes on Polyacrylamide Gels of a Deoxyribonucleic Acid-Membrane Fraction Extracted from Pneumococci

A deoxyribonucleic acid (DNA)-membrane fraction extracted from Diplococcus pneumoniae was subjected to polyacrylamide gel electrophoresis after treatment with 0.16% sodium dodecyl sulfate. At least two DNA polymerase activities were detected by in situ assays with appropriate substrates, templates, and inhibitors, including a co-polymer of deoxyadenylic and thymidylic acid and N-ethylmaleimide. This activity coincided with a fraction in the gel containing 7.5, 9.4, and 24%, respectively of the DNA, phospholipid, and protein present in the DNA-membrane fraction before electrophoresis and sodium dodecyl sulfate treatment. Assays with minced gels showed that several nuclease activities, deoxyribonucleotide kinase activity, and DNA ligase activity also coincided with this fraction. However, ribonucleoside diphosphate reductase activity did not. These results demonstrate that a complex of enzymes involved in DNA replication is firmly bound to the DNA-membrane fraction in pneumococci.     

Improved Tyrosinase Activity Stains in Polyacrylamide Electrophoresis Gels

Mammalian tyrosinase exists in a variety of subcellular locations and maturation states that result from a complex post-translational processing with possible regulatory implications. So far, SDS-PAGE has proven to be the method of choice for the resolution of tyrosinase isoforms. However, the relatively poor sensitivity of the currently available specific activity stain based on incubation of the gels with L-dopa until the formation of melanin has severely limited the use of electrophoresis in regulation studies. Two alternative staining procedures are presented and discussed. The first one involves the fluoro-graphic detection of radioactive melanin after incubation of the gels in the presence of L-[3-¹⁴C]-dopa. A similar method has already been used by others (Tsukamoto et al., 1992, Pigment Cell Res. [Suppl.] 2:84–89), but its performance has not yet been compared to the one of the dopa procedure. The sensitivity of this method can be varied by adjusting the isotopic dilution of the tracer and/or the time of exposure of the gel, but it is at least ten times higher than the one of the colorimetric stain. Moreover, the intensity of the bands is proportional to the initial tyrosinase activity over a wide range. Using this procedure, the activity present in the different subcellular fractions of melanocytes in culture can be easily detected. The second procedure involves the formation of a colored adduct between dopaquinone and MBTH. Its sensitivity is also more than one order of magnitude higher than the one obtained with L-dopa alone, and comparable to the one of the fluorographic method, but, as opposed to this latter, the complete staining can be performed in less than 1 hr.     

作用于pNPP的磷酸酶在凝胶中的特异性染色

本方法能在聚丙烯酞胺凝胶中快速,简便,灵敏和特异地染能以对硝基苯磷酸盐（pNPP）为底物的磷酸酶．它是根据Goren等人在凝胶中特异性染色环核苷酸磷酸二酯酶的方法[1]改进而成．这是基于pNPP被对硝基苯磷酸酶（pNPPase）作用后释放出的Pi在凝胶中结合铅离子形成磷酸铅,沉淀在胶中形成白色区带,再用硫化铰处理凝胶,将磷酸铅转变为硫化铅,从而使白色区带转变为棕黑色区带．它可同时分析和比较不同动物或细胞以及用不同药物处理的同一来源的动物或细胞的细胞粗提物中pNPPase的生化性质,还可在纯化此类酶的过程中,提前测定在粗提…     

Simple, Sensitive Zymogram Technique for Detection of Xylanase Activity in Polyacrylamide Gels

A method capable of detecting as little as 0.11 U of xylanase activity in polyacrylamide gels was developed. The method entails incubation of protein gels in contact with substrate gels containing unmodified xylan, followed by immersion of substrate gels in 95% ethanol. Resulting zymograms contain transparent bands corresponding to enzymatic activity against an opaque background.     

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user’s specific biological and technical needs.     

Comigration of Two Autophagosome-Associated Dehydrogenases on Two-Dimensional Polyacrylamide Gels

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (~37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (~35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-asssisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-a-hydroxysteroid dehydrogenase (3aHSD). Silver staining indicated that this 3aHSD form would selectively bind to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3aHSD demonstrated immunostaining of four 3aHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3aHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3aHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.     

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.Several improvements of the original Coomassie protocol¹ have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution², and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol³. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff''s colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol⁴. The novel aluminum-based staining in Kang''s study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.Here, we demonstrate application of Kang''s protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.Download video file.^{(128M, mp4)}     

Detection of beta-Glucosidase Activity in Polyacrylamide Gels with Esculin as Substrate

beta-Glucosidase can be located after nondenaturing polyacrylamide gel electrophoresis by incubating the gel with 0.1% esculin and 0.03% ferric chloride. The esculetin released from esculin by beta-glucosidase action reacts with ferric ion to produce a black band, corresponding to the beta-glucosidase, against the transparent background.     

A Rapid and Efficient Method for the Isolation of Proteins from Polyacrylamide Gels

A procedure is described for the rapid and efficient electrophoretic elution of protein from polyacrylamide gels which is then collected in a dialysis bag tied to the end of a tube containing the gel slices. To illustrate the method a heterogeneous preparation of alkaline phosphatase was used from which a single homogeneous component was isolated in six hours with a recovery of 86%. The eluted protein is collected in a volume which can easily be kept below 1.5 ml, thus eliminating the need for subsequent concentration. The method has also been used successfully in two other systems in which a human lung tumor-associated antigen and glycogen synthetase from yeast were isolated. Since the method utilizes a standard analytical gel electrophoresis apparatus with no modifications or accessories, it should be immediately applicable for the isolation of many different proteins from polyacrylamide gels.     

Role of Base Composition in the Electrophoresis of Microbial and Crab DNA in Polyacrylamide Gels

Intramolecular heterogeneity of eukaryotic nuclear DNA is shown by main-band and satellite DNAs. The function of the latter is uncertain, but they migrate more slowly in electrophoresis; this seems to be determined by base composition.     

Function,Structure, and Stability of Enzymes Confined in Agarose Gels

Research over the past few decades has attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior, as the average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel-encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices.     

Electrophoretic Analysis of Ribosomal and Viral Ribonucleic Acids with a Simple Technique for Slicing Low-Concentration Polyacrylamide Gels

The electrophoretic mobilities of ribosomal ribonucleic acids (RNA) from cultured mammalian (HeLa, Vero, MDBK), avian (chick embryo), and bacterial (Escherichia coli) cells, and RNA species extracted from selected viruses (Sindbis, polio, tobacco mosaic, Sendai) were compared, employing a simple, inexpensive technique for slicing low-concentration polyacrylamide gels. The procedure provides for rapid fractionation of gels used for characterization of RNA, incorporating extrusion and serial sectioning of frozen gels. Among 28S ribosomal RNA species, Vero and MDBK were indistinguishable, whereas HeLA RNA had a slightly lower mobility (higher apparent molecular weight) and chick RNA had a higher mobility (lower apparent molecular weight). The 18S ribosomal RNA species of the three mammalian sources were indistinguishable, but chick 18S RNA had a slightly lower apparent molecular weight. The inverse relation between mobility and log-molecular weight among the ribosomal and viral RNA species, though not highly precise, demonstrates the applicability of the technique to the study of molecular weights of viral RNA species.     

Identification of Neuron-Specific Enolase and Nonneuronal Enolase in Human and Rat Brain on Two-Dimensional Polyacrylamide Gels

The location of the enzymes neuron-specific enolase and nonneuronal enolase on two-dimensional gels generated from tissue samples obtained from fresh human and rat cortex has been identified. This identification is based upon the following criteria: comigration on polyacrylamide gels with the appropriate purified protein and staining on nitrocellulose protein blots of human and rat cortex using antibodies specific for each protein. The results show that our preparation of neuron-specific enolase from rat and human brain is highly pure, as only one spot is obtained on two-dimensional gels. Further, the antiserum to neuron-specific enolase is highly specific, as it reacts only with neuron-specific enolase on nitrocellulose blots derived from two-dimensional gels of cortical tissue. The location of these proteins is of interest because it positively identifies two major brain proteins on two-dimensional polyacrylamide gels of fresh cortical tissue. This information will be useful in a variety of future studies aimed at both identifying specific proteins on two-dimensional gels and observing the effects of experimental manipulations on brain and other neuronal proteins.