Detection of cytochromes on sodium dodecylsulphate-polyacrylamide gels by their intrinsic fluorescence

Heme-containing polypeptides can be detected on sodium dodecylsulphate (SDS)-polyacrylamide gels by the intrinsic fluorescence of their prosthetic group. The fluorescence is stable and specific; approximately 0.2 μg of cytochrome c/band can be detected in this way. No equipment is necessary beyond a camera, an ultraviolet lamp, and some filters.     

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.     

Visualization of protein-SDS complexes in polyacrylamide gels by chilling

A method is described which gives direct visualization of protein bands in sodium dodecyl sulfate electrophoresis gels. The procedure consists of chilling the gels to 0–4°C and observing the white opaque bands which correspond to the stained protein bands. Ovalbumin can be detected by this method at a minimum concentration of 0.2 μg per mm² of gel surface area.     

Two-dimensional zymogram analysis of nucleases in Bacillus subtilis

A two-dimensional zymogram procedure for the analysis of nucleases is described. Isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) were compared as first dimensions in combination with sodium dodecyl sulfate (SDS) electrophoresis as the second dimension in analyzing nucleases in lysates of Bacillus subtilis. All renaturable nucleases detected following SDS electrophoresis alone were resolved in NEPHGE-SDS electrophoresis gels whereas, in IEF gels, most either were at the basic end or were not present in the second-dimension gels. This method of analysis has revealed a complexity in nuclease species in B. subtilis not previously recognized. Eighty-three discreet nuclease activities have been detected in B. subtilis lysates. Using purified deoxyribonuclease I (bovine pancreas), as little as 10 pg of nuclease can be detected.     

An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels

The use of 3,3′,5,5′-tetramethylbenzidine-H₂O₂ as a stain for the peroxidase activity of cytochrome P-450 (or cytochrome P-450 in sodium dodecyl sulfate polyacrylamide gels is described in this report. This reagent can be used to detect very low levels of heme-associated peroxidase activity. The blue-stained bands on polyacrylamide gels are distinet, and the color is stable. The stained gels can be photographed or scanned at 690 nm because the gel background remains clear. The stain is easily removed from the gels to permit subsequent protein staining. Staining first for peroxidase activity has no effect on the subsequent protein staining profile. The peroxidase activity of cytochrome P-450 (or cytochrome P-420) in immunoprecipitates in Ouchterlony double diffusion plates can also be detected using this reagent.     

Staining of alkali-labile phosphoproteins and alkaline phosphatases on polyacrylamide gels

A sensitive staining method for alkali-labile phosphoproteins has been developed. As little as 0.2 nmol bound P/mm2 can be detected. The procedure is based on alkaline hydrolysis, phosphate capture, and formation of an insoluble rhodamine B-phosphomolybdate complex. A further modification for the qualitative detection of alkaline phosphatase activity on polyacrylamide gels is proposed. During incubation, the released Pi is precipitated as lead phosphate and subsequently stained with rhodamine B.     

A simple and sensitive staining method for the detection of cellulase isozymes in polyacrylamide gels.

A simple and rapid staining procedure is described for qualitative and quantitative determination of the activity of plant (Citrus sinensis (L.) Osbeck cv. Shamouti) and fungal (Trichodermata viride) cellulases in polyacrylamide gels. The method is based on the incorporation of carboxymethyl cellulose, a cellulase substrate, into the gels. After electrophoresis of crude extracts the gels are incubated in sodium-potassium phosphate buffer for the cellulase reaction which is stopped at the desired time by acidification of the gels in 60% sulfuric acid. The gels are then exposed to 2.0% KI + 0.2% I₂. No color develops in areas containing cellulase activity. The experimental procedure is described, and its different aspects are discussed.     

Detection of proteolytic enzymes in polyacrylamide gels

Aminopeptidases (1), dipeptidyl aminopeptidases (2), pyrrolidonyl peptidases (3), and carboxypeptidases (4,5) can be detected in polyacrylamide gels with appropriate-β-naphthylamide or carbonaphthoxyamino acid substrates while dipeptidases, tripeptidases (6), carboxypeptidases (7), and aminopeptidases can be detected by the coupled l-amino acid oxidase-peroxidase method of Lewis and Harris (6).In contrast, fewer methods are available for the detection of proteinases in gels. Trypsin-like (8,9) and chymotrypsin-like (5,10) proteinases can be detected with chromogenic β-naphthylamide and β-naphthol ester substrates, but proteinases such as thermolysin (11) and other bacterial neutral metal chelator-sensitive proteinases (12) cannot. For these latter proteinases, whose specificities are directed towards the amino acid residue containing the amino group of the bond to be hydrolyzed, and for proteinases, whose specificities remain to be determined, other methods of detection have to be employed.Uriel and Avrameas (13) detected proteinases in agarose gels by overlaying these gels with a second agarose gel mixture containing the substrate and a suitable pH indicator. However, the method suffers from interference by gel buffers and the instability of the pattern developed. Another procedure is to bring the gel in contact with a gelatinous layer of film material (14,15). This has been done successfully with tissue sections (16), paper electrophoretograms (17) and agarose gel separations (18).The most suitable approach is to diffuse an appropriate protein substrate into the gel after electrophoresis and detect the proteinase activity directly. Several variations of this method have been published (19–22), each with its own advantages and disadvantages. In this report a simple, sensitive method using cytochrome c as substrate, and requiring no staining, is described. This report describes its application to the detection of thermolysin and trypsin in anionic and cationic gel systems, respectively. The method has also been routinely used to locate bacterial and insect proteinases after electrophoresis.     

Unravelling the differences: comparative proteomic analysis of a clonal virulent and an attenuated Histomonas meleagridis strain

The current study focused on Histomonas meleagridis, a unicellular protozoan, responsible for histomonosis in poultry. Recently, the occurrence of the disease increased due to the ban of effective chemotherapeutic drugs. Basic questions regarding the molecular biology, virulence mechanisms or even life cycle of the flagellate are still puzzling. In order to address some of these issues, we conducted a comparative proteomic analysis of a virulent and an attenuated H. meleagridis strain traced back to a single cell and propagated in vitro as monoxenic mono-eukaryotic cultures. Using two-dimensional electrophoresis (2-DE) for proteome visualization with computational 2-DE gel image and statistical analysis, upregulated proteins in either of the two H. meleagridis strains were detected. Statistical analysis fulfilling two criteria (≥threefold upregulation and P?<?0.05) revealed 119 differentially expressed protein spots out of which 62 spots were noticed in gels with proteins from the virulent and 57 spots in gels with proteins from the attenuated culture. Mass spectrometric analysis of 32 protein spots upregulated in gels of the virulent strain identified 17 as H. meleagridis-specific. The identification revealed that these spots belonged to eight different proteins, with the majority related to cellular stress management. Two ubiquitous cellular proteins, actin and enolase, were upregulated in multiple gel positions in this strain, indicating either post-translational modification or truncation, or even both. Additionally, a known virulence factor named legumain cysteine peptidase was also detected. In contrast to this, mass spectrometric analysis of 49 protein spots, upregulated in gels of the attenuated strain, singled out 32 spots as specific for the flagellate. These spots were shown to correspond to 24 different proteins that reflect the increased metabolism, in vitro adaptation of the parasite, and amoeboid morphology. In addition to H. meleagridis proteins, the analysis identified differential expression of Escherichia coli DH5α proteins that could have been influenced by the co-cultivated H. meleagridis strain, indicating a reciprocal interaction of these two organisms during monoxenic cultivation.     

Entamoeba invadens, encystation process and enolase

The reptilian parasite Entamoeba invadens is accepted as a model for the study of the Entamoeba encystation process. Here we describe the production and characterization of a mAb (B4F2), generated against a component of the E. invadens cyst wall. This mAb specifically recognizes a 48-kDa protein present in cytoplasmic vesicles of cells encysting for 24 h. In mature cysts (96 h), the antigen was detected on the cyst surface. By two-dimensional electrophoresis and mass spectrometry analysis, the B4F2 specific antigen was identified as enolase. Levels of enolase mRNA were increased in encysting cells and the B4F2 mAb was found to inhibit cyst formation. Therefore, these results strongly suggest a new role for enolase in E. invadens encystation, and the B4F2 mAb will be useful tool to study its role in the differentiation process.     

Purification and characterization of enolase fromEscherichia coli

A method for the purification of enolase (EC 4.2.1.11) from an overproducing strain ofEscherichia coli JA 200 pLC 11–8 is described. The procedure included treatment of the crude sonic extract with protamine sulfate, followed by ammonium sulfate fractionation, hydrophobic interaction chromatography with phenyl Sepharose, HPLC ion exchange chromatography with a DuPont Sax column, and HPLC hydrophobic interaction chromatography with a Bio-Rad 5-PW column. The enzyme was purified to homogeneity as determined by silver staining of 10% sodium dodecylsulfate polyacrylamide gels. The native molecular weight ofE. coli enolase was found to be 92 kilodaltons and consisted of two subunits of identical molecular weight, 46 kilodaltons each. The isoelectric point was found to be 4.9.     

Visualization by chilling of protein bands in polyacrylamide gels containing 8 M urea: preparation and quantitation of foot-and-mouth disease virus capsid proteins

Protein bands become visible in polyacrylamide gels containing 8 m urea after chilling the gels in air for 5 to 10 min at ?70°C. Urea appears to crystallize preferentially as opaque bands in regions of the gel where protein reduces the amount of free water available as solvent for the urea molecules. Thus detected, the gel sections containing protein bands from foot-and-mouth disease virus can be immediately cut out, and their proteins obtained by electrophoretic elution or extraction procedures. Analysis of the proteins for purity and concentration is then carried out by electrophoresing measured aliquots on analytical gels, staining with Coomassie brilliant blue, scanning the gels for absorbance at 600 nm, and converting peak areas to micrograms of protein using Folin phenol standard curves determined for each purified capsid protein. The most basic capsid protein and its in virion proteolytic-cleavage products stain metachromatically.     

Plant Nucleases: III. Polyacrylamide Gel Electrophoresis of Corn Ribonuclease Isoenzymes

Isoenzymes of RNase were detected in plant extracts after polyacrylamide gel electrophoresis with a new buffer system. The gels were incubated in an RNA solution, then dipped for 30 seconds into 0.2% toluidine blue. The method is rapid and is sensitive to very small amounts of RNase. The effects of buffers and ethylenediaminetetraacetate on the different enzymes are illustrated by photographs and scans of the gels.     

A soybean seed urease-null produces urease in cell culture

Itachi, a soybean (Glycine max [L.] Merr.) variety with 0.2% normal seed urease activity, was recovered from a screen of 6,000 entries in the United States Department of Agriculture soybean germplasm collection. No urease antigen in Itachi seed extracts was detected by double diffusion or by rocket immunoelectrophoresis. Native gels stained for protein or ureolytic activity revealed no detectable urease holoenzyme. An anti-urease antibody affinity column was used to remove all detectable urease activity and antigen from `wild type' (cv. Prize) seed extracts. Affinity column effluent and nonchromatographed Itachi extracts both lack a species which comigrates with purified urease subunits in sodium dodecylsulfate polyacrylamide gels. Inability to detect urease antigen or urease protein suggests that during development of Itachi seeds there is no synthesis of urease protein or that, at most, its synthesis is 0.2% of wild type (Prize).     

Rapid alkaline blot-transfer of viral dsRNAs

The double-stranded genomic RNAs of reovirus and bluetongue virus can be transferred very efficiently from either sodium dodecyl sulfate-polyacrylamide gels or NuSieve agarose gels onto several nylon membranes. After a brief acid depurination treatment, viral dsRNAs from the gels are transferred at room temperature using 0.2 N NaOH as the transfer medium. Four blots can be obtained within 1 h and each blot contains 15-20% of the input RNA sample. These blots can be used immediately without baking in vacuo. Less than 5% of the "fixed" dsRNAs are removed after repeated washings of the membrane blots. As little as 10 pg of the genomic dsRNA segment can be detected in this alkaline Northern blot. A 20- to 50-fold increase in resolution and sensitivity over traditional Northern blots is routinely achieved. These alkaline blots can be reused 6-10 times after appropriate strip washing and proper handling.     

The tritium labeling of small amounts of protein for analysis by electrophoresis on sodium dodecyl sulfate--polyacrylamide slab gels.

A simple and reproducible method for the tritium labeling of small amounts of proteins prior to analysis under denaturing conditions on polyacrylamide slab gels is described. The method involves the in vitro labeling of proteins by reductive methylation using formaldehyde and high specific activity [³H]potassium borohydride. Labeled proteins were detected by fluorography after fractionation on polyacrylamide slab gels in the presence of sodium dodecylsulfate. The overall procedure allows the analysis and molecular weight estimation of submicrogram quantities of protein.     

Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 2-aminoacridone: subpicomolar detection using an imaging system based on a cooled charge-coupled device.

Numerous monosaccharides and oligosaccharides were derivatized at their reducing end groups with the fluorophore 2-aminoacridone. The resulting fluorescent compounds were separated by PAGE using two different buffer systems. One of these, a Tris borate buffer, enabled all of the fluorescent saccharide derivatives tested to be electrophoresed and various positional isomers, anomers, and epimers could be separated. The other system consisted of a discontinuous Tris-HCl/Tris-glycine buffer and enabled the electrophoresis of acidic, but not neutral, saccharide derivatives. The acidic and neutral saccharides could thus be distinguished unequivocally. The fluorescent labeling procedure was virtually quantitative and as little as 0.63 pmol could be detected photographically when gels were illuminated by uv light. When gels were viewed using an imaging system based on a cooled charge-coupled device, as little as 0.2 pmol was detected. The method may be useful for the structural analysis of the carbohydrates of glycoconjugates and other naturally occurring oligosaccharides.     

Enolase isoenzymes as markers of differentiation in teratocarcinoma cells and normal tissues of mouse

The changing profile of enolase (EC 4.2.1.11) isoenzymes in differentiating mouse cells has been traced by the use of specific antisera to the three subunits α, β, and γ. The amounts of the isoenzymes were measured in a variety of tissues during normal mouse development and during the differentiation of mouse teratocarcinoma cells in culture and as tumors. One isoenzyme is predominant in the early cells of the developing mouse embryo, namely, the homodimer made up of α subunits. The same isoenzyme is also the sole form detected in undifferentiated teratocarcinoma (embryonal carcinoma) cells. The isoenzyme form remains unchanged in developing primitive and definitive endoderm of the embryo. Similarly, endoderm cells formed by differentiation of embryonal carcinoma cells contained only αα enolase. In contrast, during the development of striated muscle and of brain, increasing proportions of β and γ subunits, respectively, were detected. Thus enolase was found to be a marker of the differentiation of these tissues. This conclusion was substantiated by finding significant amounts of the β subunit in teratocarcinoma cell cultures which had formed beating striated muscle in vitro.     

Selective elution of zones from preparative isoelectric focusing gels by ampholytes or buffers.

Protein zones formed by isoelectric focusing on polyacrylamide gels (IFPA) can be eluted without mechanical disruption of the gels. Specific elution is achieved by replacement of the original anolyte, a strong acid, with an ampholyte of a pI higher than that of the protein which is to be eluted. Alternatively, the anolyte may be a buffer of a pH higher than the pI of the focused protein zone. A rudimentary apparatus and procedures for the application of this method of zone elution are described but are not as yet sufficiently developed to provide a ready-to-use preparative IFPA procedure.