Activity assay for nisin-like acidic bacteriocins using an optimal pH-conditioned gel matrix

A new zymography for detecting nisin-like acidic bacteriocins was developed using a tricine-sodium dodecyl sulfate (SDS) gel and an acidic gel matrix (pH 4.0). After electrophoresis, proteins in the tricine gel were electrotransferred to an optimal pH-conditioned gel matrix (OP-CGM). The OP-CGM was overlaid with indicator cells (Bacillus cereus) embedded in nutrient broth soft agar (0.8%, w/v). Antibacterial activity shown as a growth inhibition using B. cereus was detected at approximately 3.8 kDa. Because nisin is unstable in buffers at pH values over 6.0, the common electrophoretic systems, SDS-polyacrylamide gel electrophoresis and tricine gel, are not suitable for detection of nisin-like acidic bacteriocins.     

Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel

This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing β-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.     

Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel

We have developed a system to detect three hydrolytic enzymes (cellulase, lipase, and protease) using a single sodium dodecyl sulfate (SDS) gel and an electrotransfer system. After electrophoresis, proteins in the gel were transferred to three sandwiched substrate gels containing glycerol tributyrate, azo-carboxymethyl cellulose (Azo-CMC), and fibrin for detection of cellulase, lipase, and protease, respectively. We show that three cellulases (from a Paenibacillus sp. and two Bacillus sp. strains), one lipase (from a Staphylococcus sp.), and two proteases (from two Bacillus sp. strains) can be detected simultaneously with our zymogram system.     

Purification and identification of a Bacillus nitroreductase: Potential use in 3,5-DNBTF biosensoring system

We have investigated a specific enzymatic biosensor for detecting target pollutant 3,5-dinitro-trifluoromethylbenzene (3,5-DNBTF). The predicted enzyme is a nitroreductase that catalyzes the total nitroreduction of 3,5-DNBTF to its corresponding diamine. The photo-activation of this diamine offers a large panel of detection tools. After broad screening of microorganisms, only the strains belonging to the genus Bacillus were able to reduce the two nitro groups of 3,5-DNBTF. Among them, Bacillus LMA, isolated from explosives-polluted effluents, was the most efficient in reducing this compound. The involved nitroreductase was identified by 2D gel electrophoresis coupled to mass spectrometry, as the Bacillus subtilis oxygen-insensitive nitroreductase NfrA. The enzyme was purified by mono-P chromatofocusing.     

Detection of protein complex interactions via a Blue Native-PAGE retardation assay

We describe the Blue Native (BN)-PAGE retardation assay for the detection of interactions of biomolecules with protein complexes. Potential interactors of proteins are included in the BN gel matrix, resulting in retardation of proteins that interact with the added molecule. After validation using the T-cell antigen receptor, we applied the assay for a general identification of dextran interactors in combination with mass spectroscopy. The proteomic screen revealed triosephosphate isomerase oligomer as a dextran-binding, high M_R complex.     

A method for the direct measurement of glycogen synthase activity on gels after polyacrylamide gel electrophoresis

A method for the detection of glycogen synthase activity after nondenaturing polyacrylamide gel electrophoresis is described. After the electrophoretical run, the gels were incubated in situ with UDP-glucose and glycogen. Labeled or unlabeled UDP-glucose could be used, since similar activity patterns were obtained by autoradiography or iodine staining of the gels. The method here described offers several advantages in terms of speed, sensitivity, and economy when compared with other procedures.     

Microelectrophoretic method for the detection of proteinase inhibitors

A microelectrophoretic method for the detection of proteinase inhibitors is deseribed. Microscope slides covered with agar gel containing casein were used for the electrophoretic separation of proteins from bean seeds. Subsequently, the slides were covered with a filter paper strip saturated with a proteinase solution of a known concentration. After 60 min of digestion, the filter paper strips were removed, and the slides were stained with amido black. Blue spots appeared where the casein substrate was protected from digestion by the presence of proteinase inhibitors. Single seed or leaf extracts can be studied by this method. Different trypsin inhibition patterns were observed for samples of different bean varieties. An inhibitor for subtilisin was detected in all the Ph. vulgaris samples studied.     

Development of TaqMan Probe-Based Insulated Isothermal PCR (iiPCR) for Sensitive and Specific On-Site Pathogen Detection

Insulated isothermal PCR (iiPCR), established on the basis of Ralyeigh-Bénard convection, is a rapid and low-cost platform for nucleic acid amplification. However, the method used for signal detection, namely gel electrophoresis, has limited the application of iiPCR. In this study, TaqMan probe-based iiPCR system was developed to obviate the need of post-amplification processing. This system includes an optical detection module, which was designed and integrated into the iiPCR device to detect fluorescent signals generated by the probe. TaqMan probe-iiPCR assays targeting white spot syndrome virus (WSSV) and infectious myonecrosis virus were developed for preliminary evaluation of this system. Significant elevation of fluorescent signals was detected consistently among positive iiPCR reactions in both assays, correlating with amplicon detection by gel electrophoresis analysis. After condition optimization, a threshold value of S/N (fluorescent intensity_after/fluorescent intensity_before) for positive reactions was defined for WSSV TaqMan probe-iiPCR on the basis of 20 blank reactions. WSSV TaqMan probe-iiPCR generated positive S/Ns from as low as 10¹ copies of standard DNA and lightly infected Litopenaeus vannamei. Compared with an OIE-certified nested PCR, WSSV TaqMan probe-iiPCR showed a sensitivity of 100% and a specificity of 96.67% in 120 WSSV-free or lightly infected shrimp samples. Generating positive signals specifically and sensitively, TaqMan probe-iiPCR system has a potential as a low-cost and rapid on-site diagnostics method.     

Preparation of a stable folate-sepharose complex for affinity chromatography.

A stable folic acid affinity gel has been developed for the purification of nanograms of protein that bind folic acid or its derivatives. The affinity gel was prepared by first coupling folic acid covalently to bovine serum albumin, followed by covalent coupling of the albumin to p-benzoquinone-activated Sepharose. After the albumin-folic acid complex was formed, it was treated with charcoal to remove ionically bound folate which would otherwise elute from the gel and decrease the recovery of the binding protein. The p-benzoquinone activation resulted in a more stable binding of the albumin to the Sepharose.     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

Methane- and n-butaneboronates: nw derivatives for gas-chromatographic analysis of 3-methoxy-4-hydroxyphenylethylenglycol.

Several methods were described for visualization of proteolytic activity on electrophoregrams obtained with agar, agarose, starch, or acrylamide gels as supporting media. In most of these reports casein or hemoglobin were used as nonspecific substrates (1–3). Recently, colorimetric assays for trypsin, using α-benzoyl-d,l-arginine-p-nitroanilide (4), and for subtilisin, using Z-glycyl-glycyl-l-leucine-p-nitroanilide (5) were introduced for the localization of these proteases after acetate celulose and acrylamide gel electrophoresis. No convenient and simple methods were in practice for detection of leucineaminopeptidase (3), although this enzyme was assayed in solution with specific chromogenic substrates such as l-leucine-p-nitroanilide or l-leucine-β-naphtylamide (6,7).The present report describes the use of p-nitroanilide substrate-l-leucine-p-nitroanilide for detection of leucineaminopeptidase activity after acrylamide gel electrophoresis. The method allows a rapid and sensitive localization of leucineaminopeptidases.     

Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with fungal laccase

An enzyme-based method for destaining polyacrylamide gels stained with Coomassie Brilliant Blue R-250 is described. Distilled water supplemented with diluted fermentation broth of a laccase-producing white-rot fungus, Cerrena sp., was used for gel destaining, and a clear gel background was obtained in 2 h at 37 °C. Sensitivity of protein detection was 10 ng. The method did not require organic solvents or changing the destaining solution. Due to simultaneous gel destaining and dye decolorization, the colorless destaining solution can be disposed of directly. Laccase destaining of polyacrylamide gels was simple, efficient, and environmentally friendly.     

Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis

Euglena chloroplast polypeptides are resolved by an adaptation of the two-dimensional gel electrophoretic technique of O'Farrell (1975 J Biol Chem 250: 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as well as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Euglena chloroplast polypeptides are resolved on one-dimensional sodium dodecylsulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the first dimension, sodium dodecylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Euglena chloroplasts with acetone-ether and subsequent solubilization of polypeptides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two-dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when ³⁵S-labeled chloroplast polypeptides are separated by the present two-dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, autoradiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography.     

Proteomic and Systems Biology Analysis of the Monocyte Response to Coxiella burnetii Infection

Coxiella burnetii is an obligate intracellular bacterial pathogen and the causative agent of Q fever. Chronic Q fever can produce debilitating fatigue and C. burnetii is considered a significant bioterror threat. C. burnetii occupies the monocyte phagolysosome and although prior work has explained features of the host-pathogen interaction, many aspects are still poorly understood. We have conducted a proteomic investigation of human Monomac I cells infected with the Nine Mile Phase II strain of C. burnetii and used the results as a framework for a systems biology model of the host response. Our principal methodology was multiplex differential 2D gel electrophoresis using ZDyes, a new generation of covalently linked fluorescent protein detection dyes under development at Montana State University. The 2D gel analysis facilitated the detection of changes in posttranslational modifications on intact proteins in response to infection. The systems model created from our data a framework for the design of experiments to seek a deeper understanding of the host-pathogen interactions.     

Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library

Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08–6.44 ng mL⁻¹ and the medium inhibition of control (IC₅₀) was 0.73 ng mL⁻¹. It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%–96.6% and with a coefficient of variation (CV) among 2.0%–8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples.     

Disc electrophoretic studies of hemoglobin dissociation by p-hydroxymercury benzoate

The reaction of excess p-mercurybenzoate with human hemoglobin produces five electrophoretic species on polyacrylamide gel. Only two of these bands have been previously observed when starch gel was employed. The chemical and electrophoretic studies presented in this paper illustrate that the appearance of an “extra” band in the β zone is due to the ability of PAGE to separate the βb₂^PMB ? β^PMB equilibrium to its discrete components. The remaining bands are accounted for by the superior resolution of PAGE over starch gel electrophoresis which allowed the detection of two (αβ)^PMB dimer species.     

Method of detection of protein kinase activity in polyacrylamide gel using two-dimensional protein separating system

Method for detection of protein kinase activity in polyacrylamide gel have been developed. After separation of proteins by isoelectric focusing in non-denaturing condition, gel was incubated in a reaction buffer containing [gamma-32P]ATP. 32P-labeled proteins were separated by subsequent SDS/PAGE electrophoresis in second dimension. The proposed method was used for detection of protein kinase activity in human blood serum and triton X-100 soluble proteins of heads of Drosophila melanogaster.     

In-gel protein phosphatase assay using fluorogenic substrates

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.     

Suicide Polymerase Endonuclease Restriction, a Novel Technique for Enhancing PCR Amplification of Minor DNA Templates

PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from Streptomyces spp., and selective restriction of the Streptomyces 16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA.     

Detection of proteases by clotting of casein after gel electrophoresis

Clotting of casein provides a sensitive method for detection of proteases after gel electrophoresis. The method is here designated "caseogram." After electrophoresis the gel was equilibrated with 0.15-0.3 M sodium acetate, pH 5.3, and an 1% agarose gel containing 1% skim-milk powder in 0.1 M sodium acetate, pH 5.3, was placed on top of the electrophoresis gel. By incubation at 37 degrees C for 2 h the protease-containing zones produced distinct precipitates in the skim-milk gel. For permanent documentation the skim-milk gel was stained with amido black. The detection limit for pepsin A is 5 ng in the caseogram against 25 ng by hemoglobin digestion at pH 2.5. For calf chymosin it is 1 ng against 100 ng by digestion of hemoglobin at pH 3.5. Caseograms work well after agar gel electrophoresis, after different types of immunoelectrophoresis, and after isoelectric focusing or disc electrophoresis in polyacrylamide gels. Since inert proteins do not interfere with the detection, the method is especially suitable for analysis of crude samples. Samples containing pepsinogen or pepsinogen-like zymogens may be activated at pH 2 before equilibration at pH 5.3.