Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels

A procedure is described for identifying protein kinase activity in protein samples following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Protein kinase activity is detected by renaturation of the enzymes within the gel followed by phosphorylation with [gamma-32P]ATP of either substrates included in the polyacrylamide gel or of the kinase itself. Then, after removal of the unreacted [gamma-32P]ATP by washing the gel in the presence of an anion-exchange resin, the positions (Mr) of the protein kinase activity are visualized by autoradiography. Studies using a purified catalytic subunit of cAMP-dependent protein kinase indicate that enzyme concentrations as low as 0.01 microgram can easily be detected on gels containing 1 mg/ml casein. The technique is also useful for identifying active subunits of multisubunit enzymes. The active subunit of casein kinase II, for example, can readily be determined by renaturing the dissociated enzyme in gels containing casein. Putative protein kinases present in crude mixtures of proteins can also be detected following separation by gel electrophoresis and can be characterized on the basis of molecular weight and identity of the phosphorylated amino acid. Using this technique, at least three major protein kinases were detected in a mixture of proteins prepared by subfraction of red blood cell membranes.     

A new enzymatic staining method for the detection of radish beta-fructosidase in gel electrophoresis

Optimum conditions for β-fructosidase detection in polyacrylamide and agarose gels are defined from comparison of zymograms obtained with two staining methods including an original one. Under all conditions tested in the present study detection has been improved with the new staining procedure. The new staining medium developed here uses two intermediary enzymes: glucose oxidase and peroxidase, 3.3′-diaminobenzidine as final acceptor, and sucrose as substrate for β-fructosidase. β-Fructosidase zymogram is obtained either by gel immersion in this staining solution, or when highly crosslinked polyacrylamide separating gels are used, by overlaying the slab within a thin buffered agarose gel containing staining chemicals (“sandwich technique”). Examples are presented to illustrate the general principles involved and indicate the conditions necessary for optimal development of this precise and specific technique.     

Detection of pectic enzymes in pectin-acrylamide gels

A rapid, sensitive slab gel electrophoretic method for the study of pectic enzymes is described. By incorporating pectin in acrylamide gels and subsequent staining with ruthenium red, simultaneous detection of esterases, glycosidases, and lyases was possible due to their differing staining reactions. Application of the method to the study of the thermal stability of enzymes is described. Approximate isoelectric points of the enzymes could be determined from their positions in the gel.     

An electroblotting apparatus for multiple replica technique and identification of human serum proteins on micro two-dimensional gels

A new apparatus for electrophoretic transfer of proteins from micro polyacrylamide slab gels has been developed. The apparatus enabled the easy changing of nitrocellulose sheets and was suited for obtaining multiple blots from a gel. Electrophoretic conditions were determined so that all of the blots obtained sequentially from one slab gel were successfully used to visualize specific proteins irrespective of their molecular weight. Combining the transfer technique with the technique of parallel micro two-dimensional electrophoresis, 20 blots could be obtained within 1 h of electroblotting time. The locations of 28 human serum proteins were determined simultaneously on these blots using commercial specific antisera.     

DNA sequencing separations in capillary gels on a modified commercial DNA sequencing instrument

DNA sequencing separations of standard DNA fragments of known sequence have been achieved in small diameter capillary gels electrophoresed and analyzed in parallel in a modified commercial DNA sequencer instrument. DNA sequencing in terms of base-calling accuracy is comparable to conventional slab gels; however, the separations in the capillary were performed somewhat faster and required less sample than those in the slab gel. Advantages of this approach vs. separations on conventional slab gels are discussed.     

A technique for electrophoresis in multiple-concentration agarose gels

A technique has been developed for embedding several agarose gels (running gels), each of a different agarose concentration, within a single 1.5% agarose slab. Equal portions of a sample were placed at the origin of each running gel and were simultaneously subjected to electrophoresis. Protein within the running gels was detected by staining with Coomassie blue; 0.2% gels were the least concentrated gels that were stained without gel breakage. Using the above technique, the dependence of electrophoretic mobility on agarose concentration has been measured for bacteriophage T7 capsids and a capsid dimer.     

Protein staining and pH gradient determination on the same gel in isoelectric focusing.

The apparent isoelectric point of a component focused on polyacrylamide gels is normally estimated by extrapolating a pH gradient determined on one gel to another gel which has been stained for protein in order to locate the position of the component (1). The pH gradient is determined by slicing the gel transversely and reading the pH of the eluate after soaking the segments for 1–2 hr in a small amount of degassed water. It is assumed that the gradients in both gels are identical. Alternatively, an antimony microelectrode has been used to measure pH gradients directly in unsectioned gels (2). Similar techniques have been applied to polyacrylamide gel slabs and are reviewed by Vesterberg (3). Righetti and Drysdale (4) have recently reviewed these and other aspects of isoelectric focusing in gels.I report here a very precise method for the determination of a protein “isoelectric point” that can be accomplished with a single gel. The technique is demonstrated with yeast phosphoglycerate kinase and the very low density lipoprotein (VLDL) fraction from human plasma.     

Zone precipitation chromatography: its use in the isolation of different collagen types

Zone Precipitation Chromatography is a useful technique for the initial isolation of the different collagen types in their native configuration. Small quantities of collagen mixtures can be rapidly separated into different collagen types with a relatively high degree of purity, based upon stained protein patterns on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) slab gels. In the commonly used bulk salt preparative method for isolating the different collagens, 50 mg of starting material was needed. Three days were required to complete the procedure. The stained protein patterns on SDS-PAGE slab gels showed about 25% contamination with the bulk purified Type III fraction and 20% contamination with the bulk purified type AB collagen. With Zone Precipitation Chromatography 5 mg of starting material was used and in less than 4 hours the mixture was separated with Types III and AB fractions showing less than 10% contamination from other collagen types. The technique is patterned after the Zone Precipitation method reported by Porath seventeen years ago and utilizes a step-wise sodium chloride gradient to precipitate and redissolve the collagens, eluting from the interbead spaces of a molecular sieve column.     

In-gel precipitation of enzymatically released phosphate

The phosphate precipitation reaction using ammonium molybdate and triethylamine under low pH has been applied to gel-based assays for detecting phosphate-releasing enzymes. The sensitivity of the assay is 10 pmol Pi/mm2 of 1.5-mm-thick gel. The assay is applicable to enzymes with a wide range of optimal pH, from acid (pH 4.5) to alkaline phosphatase (pH 9.7), and to enzymes that use acid-labile substrates such as apyrase and glutamine synthetase. Using a negative staining approach, maltose phosphorylase, a phosphate-consuming enzyme, can also be detected. The assay was used to detect glutamine synthetase isoforms, separated by nondenaturing polyacrylamide gel electrophoresis from crude maize extracts. For downstream applications such as staining gels for proteins, the gels with precipitate should be incubated in 10 mM dithiothreitol or beta-mercaptoethanol until the precipitate is dissolved and then thoroughly washed in water. In comparison to calcium phosphate precipitation or the phosphomolybdate-malachite green method, this method is more sensitive. It is a very simple, rapid, versatile, reproducible, and inexpensive method that could be a useful tool in enzymological studies.     

Substrate-containing gel electrophoresis: sensitive detection of amylolytic, nucleolytic, and proteolytic enzymes

Electrophoresis of hydrolytic enzymes under nondenaturing conditions on acrylamide gels containing the appropriate high-molecular-weight substrates entrapped on the gel has been explored as a general method for sensitive enzyme resolution and detection. Under electrophoresis conditions of optimal enzyme activity, the enzymes may bind tightly to the fixed substrate and can only migrate in the electrophoretic field as the substrate is hydrolyzed. When the gels after electrophoresis in this “binding mode” are stained with substrate-detecting reagents, clear tracks of enzyme migration are observed, and the length of each track is a function of the amount of enzyme present in that track. Multiple forms of a given enzyme activity have not been and are not likely to be observed under these conditions. Under electrophoresis conditions of minimal (or suboptimal) enzyme activity, the enzymes do not bind to the fixed substrate and their mobility in the electrophoretic field does not appear to be significantly affected by the presence of substrate. After electrophoresis in this “nonbinding mode” the gels are incubated under conditions of optimal enzyme activity to allow substrate hydrolysis to take place before they are stained with substrate-detecting reagents, and active enzymes are detected as clear bands. Multiple forms of a given activity which were resolved during electrophoresis in the nonbinding mode are reflected by the presence of individual bands. The substrate-containing gel electrophoresis technique does not appear to be amenable to precise quantification of enzymes. By comparing the length of the clear tracks or the degree of staining of the activity bands for a range of enzyme concentrations, however, it is possible to establish the smallest amount of enzyme that can unequivocally be detected under a given set of conditions; from such studies we estimate that the sensitivity of detection with the substrate-containing gel electrophoresis technique can be orders of magnitude better than that obtained with other methods. The levels of detection observed in the work presented here were about 50 pg for α-amylase run on starch-containing gels, 1 pg to 1 ng for nucleases run on DNA- or RNA-containing gels, and 100 pg to 10 ng for 11 different pure and crude protease preparations run on gels containing heat-denatured bovine serum albumin.     

Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis

We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.     

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.     

The product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

A method termed "product-selective" blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase (EC 3.5.1.2) or glutamate synthase (EC 1.4.7.1) reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing "image gel." Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application.     

A procedure for the immunoblotting of proteins separated on isoelectric focusing gels

A method has been devised for performing Western blot assays on proteins resolved by isoelectric focusing. Electrophoretic transfer of proteins directly from isoelectric focusing (IEF) tube gels to nitrocellulose sheets allowed their immunoassay without conventional second dimension SDS gel electrophoresis. The same method can also be used for IEF slab gels. For the immunostaining of nonmuscle actin isoforms in extracts of cultured cells, the resolution of this technique was much improved over that of Western blots of two-dimensional gels.     

Modification of commerical and custom-built slab gel electrophoresis units for two-dimensional polyacrylamide gel electrophoresis

Many commercial and custom-built slab gel electrophoresis units can be modified to function as two-dimensional polyacrylamide gel electrophoresis units with the insertion of Plexiglas adapters. These adapters can be made for about $50 a pair and can be used for either temporary or permanent modification of the slab gel units. The physical dimensions of the adapters can be varied to permit great flexibility in the diameter of cylinder gels and the thickness of slab gels that can be run together. For example, proteins from 6-mm cylinder gels can be easily separated on 1-mm slab gels, which can then be dried for autoradiography.     

秦巴山区巨缘蝽亚科三种昆虫同工酶的初步研究(半翅目:缘蝽科)

为对缘蝽科分类提供生物化学和遗传数据,本研究运用聚丙烯酰胺垂直板电泳技术对巨缘蝽亚科三个种的酯酶同工酶进行了检测。结果表明:斑背安缘蝽Anoplocnemis binotataDistant不同组织酯酶酶谱不同,可能与适应特殊生理功能相关。斑背安缘蝽与波赫缘蝽Ochrochira potaniniKiritshenko、月肩奇缘蝽Derepteryx lu-nate(Distant)之间明显的遗传差异说明系统发育关系较远。三种缘蝽酯酶同工酶具有遗传多态现象,控制酯酶的基因位点均为5个,而且酯酶均为单体酶。     

A simple and efficient method for the separation and detection of small DNA fragments by electrophoresis in formamide containing agarose gels and Southern blotting to DBM-paper.

We show in this report that DNA fragments smaller than 300 bp are separated with high resolution by electrophoresis in concentrated (up to 7%) agarose gels containing 50% formamide. The separated DNA fragments can subsequently be quantitatively transferred to DBM-paper [Alwine, J.C. et al., Proc. Natl. Acad. Sci. USA (1977) 74, 5350-54] using the Southern technique [Southern, E.M., J. Mol. Biol. (1975) 98, 503-517], while preserving the sharpness of the original gel pattern. Since thin (0.2-0.4 mm) and thick (up to 5 mm) agarose slab gels can be easily handled in vertical or horizontal apparatus, this method should prove to be a very useful extention of the Southern technique, applicable to a variety of analytical and preparative purposes.     

Analytical techniques for cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient-slab gel electrophoresis

Two-dimensional electrophoresis of proteins and protein subunits, employing isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate in the second, yields the highest resolutions currently available. In this paper separations in the second dimension are considered (the so called DALT system). Methods for multiple-parallel casting of gradient gels in slab gel holders are described. The problem of electrical isolation of the ends of the slabs together with continuous cooling of both surfaces of the slab gel holder along their entire length has been achieved by running the gels in a horizontal direction in a three-compartment tank with the holders inserted in insulating septa. In the system described, 10 slabs are run simultaneously. This, however, is not the upper limit of the number of slabs which can be conveniently run in parallel.     

Biochemical Identification of the Two Races of Radopholus similis by Polyacrylamide Gel Electrophoresis

Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as diagnostic. Resolution of protein bands was greatly improved by SDS and gradient slab gel electrophoresis, but no differences could be detected among the proteins resolved between the two rares with these techniques. Two-dimensional gels revealed a large number of proteins, but background staining obscured them hindering interpretation. When nematode races were reared on three different host plants, no differences in protein patterns were detected between them, indicating host preferences does not play a role in determining the types proteins occurring in these nematodes.     

Rapid microsatellite analysis using discontinuous polyacrylamide gel electrophoresis.

A method for screening large numbers of samples for microsatellites using discontinuous, non-denaturing polyacrylamide gels and rapid fluorescent gel staining is described. Disc electrophoresis on slab gels provides high-resolution of PCR products. It is useful for collecting population data once microsatellite loci have been characterized.