Handbook of Detection of Enzymes on Electrophoretic Gels

Manchenko, Gennady P.

doi:10.1201/9781420040531

Cited by 257 publications

(216 citation statements)

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“…Efforts to visualize the enzyme after electroblotting to PVDF membranes were not successful, however. Naphthol-AS-BI b-D-glucuronide coupled to Fast Garnet GBC or 6-bromo-2-naphthyl b-D-glucuronide coupled to Fast Blue B are two color generating systems that have been used for the detection of b-glucuronidase activity directly in gels [25]. Gels are incubated in the dark in the stain solution until colored bands appear and then fixed in 7% acetic acid.…”

Section: Discussionmentioning

confidence: 99%

“…Gels are stained in a similar manner as the other chromogenic stains but must be fixed in 10% trichloroacetic acid for 8 h before storage in 7% acetic acid. This substrate may be coupled to nitroblue tetrazolium for simultaneous formation of blue dehydroindigo dye and blue formazan at the site of enzyme activity [25,26]. Detecting absorbance of these three chromogenic stains is limited by the molar extinction coefficient of their respective products, however, and we are not aware of any methods for simultaneous visualization of b-glucuronidase activity and total protein expression using the cited substrates.…”

Section: Discussionmentioning

confidence: 99%

“…The fluorogenic substrate 4-methylumbelliferyl b-glucuronide is most commonly used to measure b-glucuronidase activity [25]. In zymography, the substrate is applied to the gel surface imbibed in a filter paper and after incubating at 37 7C for 90 min, fluorescent bands are visualized using long wavelength ultraviolet (UV-A) illumination and a 420 nm long-pass emission filter (e.g.…”

Section: Discussionmentioning

confidence: 99%

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Simultaneous, two-color fluorescence detection of total protein profiles and β-glucuronidase activity in polyacrylamide gel

Kemper¹,

Steinberg²,

Jones³

et al. 2001

Electrophoresis

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A dichromatic method for measuring the specific activity of beta-glucuronidase from complex cell homogenates or partially purified protein fractions is presented. Dual fluorescence is achieved by using the green emitting fluorogenic substrate ELF 97 beta-D-glucuronide to detect beta-glucuronidase activity, followed by the red emitting SYPRO Ruby protein gel stain or SYPRO Ruby IEF gel stain to detect the remaining proteins in the electrophoretic profile. Both ELF 97 alcohol, the highly fluorescent hydrolytic product generated from the enzyme substrate, and the SYPRO Ruby total protein stains are maximally excited by ultraviolet illumination. ELF 97 alcohol emits maximally at 525 nm while the SYPRO Ruby dyes emit maximally at 610 nm. Since ELF 97 beta-glucuronide is a precipitating substrate, it allows precise localization of beta-glucuronidase activity with minimal band diffusion. The staining method is simple and direct, without the requirement for ancillary coupling reactions. Dichromatic protein detection is demonstrated after sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, carrier ampholyte-mediated isoelectric focusing or two-dimensional gel electrophoresis.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Simultaneous, two-color fluorescence detection of total protein profiles and β-glucuronidase activity in polyacrylamide gel

Kemper¹,

Steinberg²,

Jones³

et al. 2001

Electrophoresis

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“…One unit of SOD activity was deWned as the amount of enzyme which causes 50% of maximum inhibition of NBT reduction [20,22]. The SOD will show a grey band under the blue background in the nondenaturing PAGE gel after staining in the PMS-MTT solution in daylight for half an hour [9]. The protein concentration was determined using the Coomassie (Bradford) Protein Assay Kit (Pierce).…”

Section: Crude Enzyme Preparationmentioning

confidence: 99%

Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp. XMH10

Wang

Yang

Ruan

et al. 2007

J Ind Microbiol Biotechnol

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A superoxide dismutase (SOD) gene was cloned from the thermophilic bacterium Rhodothermus sp. XMH10 for the first time and highly expressed in Escherichia coli. The Rhodothermus sp. XMH10 SOD (RhSOD) gene encodes 209 amino acids with a putative molecular weight of 23.6 kDa and a pI value of 5.53. The recombinant RhSOD was detected to be an iron type SOD and existed as a dimer on its natural status. Experiments revealed that this RhSOD showed high activity at 50-70 degrees C and pH 5.0. Compared to SODs from other thermophiles, it was highly thermostable, maintaining more than 90% of its activity after incubation at 70 degrees C for 12 h, only totally inactivated after more than 4-h incubation at 80 degrees C. It also showed much higher resistance to KCN, NaN(3) and H(2)O(2) as compared to other SODs.

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“…Leucine aminopeptidase activity in the gel was detected with the procedure described by Manchenko [14]. PAGE was performed in a vertical mini-gel system (Mini-Protean III system; Bio-Rad Laboratories, Richmond, Calif., USA) with a 10% non-denaturing polyacrylamide gel.…”

Section: Expression and Purification Of Lapsbdmentioning

confidence: 99%

Fusion of Bacillus stearothermophilus leucine aminopeptidase II with the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity

Hua

Chi

et al. 2004

J IND MICROBIOL BIOTECHNOL

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Bacillus stearothermophilus leucine aminopeptidase II (LAPII) was fused at its C-terminal end with the raw-starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase. The chimeric enzyme (LAPsbd), with an apparent molecular mass of approximately 61 kDa, was overexpressed in IPTG-induced Escherichia coli cells and purified to homogeneity by nickel-chelate chromatography. The purified enzyme retained LAP activity and adsorbed raw starch. LAPsbd was stable at 70 degrees C for 10 min, while the activity of wild-type enzyme was completely abolished under the same environmental condition. Compared with the wild-type enzyme, the twofold increase in the catalytic efficiency for LAPsbd was due to a 218% increase in the k(cat) value.

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Handbook of Detection of Enzymes on Electrophoretic Gels

Cited by 257 publications

References 0 publications

Simultaneous, two-color fluorescence detection of total protein profiles and β-glucuronidase activity in polyacrylamide gel

Simultaneous, two-color fluorescence detection of total protein profiles and β-glucuronidase activity in polyacrylamide gel

Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp. XMH10

Fusion of Bacillus stearothermophilus leucine aminopeptidase II with the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity

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