Zymography methods for visualizing hydrolytic enzymes

Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Steen, Philippe E. Van den; Opdenakker, Ghislain

doi:10.1038/nmeth.2371

Cited by 261 publications

(246 citation statements)

References 72 publications

Supporting

Mentioning

236

Contrasting

Unclassified

Order By: Relevance

“…Images were obtained using the Gel Image Formation system (LI-COR Biosciences, Lincoln, NE, USA) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Pro-MMP-9 and activated gelatinase exhibit gelatinolytic activity in gelatin zymography (16). Thus, the samples were separated by SDS-PAGE (0.1% gelatin).…”

Section: Determination Of Intracellular Ph Valuesmentioning

confidence: 99%

Cleistanthin A inhibits the invasion and metastasis of human melanoma cells by inhibiting the expression of matrix metallopeptidase‑2 and ‑9

Pan

Cai

et al. 2017

Oncol Lett

View full text Add to dashboard Cite

Abstract. It has been demonstrated that numerous types of metastatic cancer overexpress vacuolar-type H + (V)-ATPases. It may be possible to inhibit the growth and metastasis of human cancer cells by inhibiting V-ATPases. It was previously reported that diphyllin, a novel V-ATPase inhibitor, can inhibit the migration and invasion of SGC7901 human gastric cancer cells; however, the effects of cleistanthin A (CA), a diphyllin glycoside, on melanoma cells has not been demonstrated. The present study aimed to investigate the effect of CA as a V-ATPase inhibitor and its effects on the invasion and metastasis of A375 cells. The results of an MTT assay in the present study indicated that the growth inhibition of A375 cells by CA was induced in a dose-and time-dependent manner; however, A375 cell viability was not significantly affected by low concentrations (0.03, 0.1 and 0.3 µM) after 24 h. Similar results were obtained by viable cell counting with trypan blue. Therefore, these concentrations of CA were selected for the treatment of A375 cells in further experiments. It was demonstrated that CA inhibited the expression of V-ATPases in a dose-dependent manner and decreased the internal pH level of A375 cells. Alterations to the lysosomal pH were associated with the CA concentration. Furthermore, CA treatment induced a significant decrease in cell migration and invasion, as demonstrated with wound-healing and Transwell assays. Gelatin zymography and western blot analysis demonstrated that the expression levels of matrix metallopeptidase (MMP)-2 and -9 decreased following CA treatment. Therefore, CA can be characterized as a novel V-ATPase inhibitor for the treatment of melanoma that may inhibit invasion and metastasis by downregulating the expression of MMP-2 and -9.

show abstract

Section: Determination Of Intracellular Ph Valuesmentioning

confidence: 99%

Cleistanthin A inhibits the invasion and metastasis of human melanoma cells by inhibiting the expression of matrix metallopeptidase‑2 and ‑9

Pan

Cai

et al. 2017

Oncol Lett

View full text Add to dashboard Cite

show abstract

“…The visualization of activity can be accomplished either by the disappearance of the substrate or the appearance of a degradation product. Zymography can be used in electrophoresis gels (in-gel zymography), tissue samples (in situ zymography) and in intact organs (in vivo zymography) [140].…”

Section: Zymographymentioning

confidence: 99%

“…The enzyme´s substrate can either be incorporated in the gel-matrix, added in a staining buffer or the gel can be blotted on membranes and overlaid with agarose containing the substrate [140]. After electrophoresis, the SDS and possible reducing agents need to be removed to allow the enzymes to refold to a hydrolytic active form, which is usually achieved by extensive washing steps using an appropriate buffer.…”

Section: Zymographymentioning

confidence: 99%

Methods development for metaproteomics-guided bioprospecting of novel enzymes

Speda¹

View full text Add to dashboard Cite

Cover: 2-D DIGE gel used for the accurate identification of extracellular cellulolytic enzymes secreted by a methanogenic microbial community. Red protein spots indicate proteins upregulated by the addition of cellulose.During the course of the research underlying this thesis, Jutta Speda was enrolled in Forum Scientium, a multidisciplinary doctoral programme at Linköping University, Sweden. Printed by LiU-Tryck, Linköping, Sweden, 2017 Look deep into nature, and then you will understand everything better. Albert Einstein v AbstractIndustrial biotechnology has been announced by several organizations and governments as a key enabling technology for the enhanced economic growth in a low-carbon and knowledge-based bioeconomy. An important goal to promote an environment friendly and sustainable industrial biotechnology is the discovery of new enzymes.To date, almost all enzymes used in industry have been discovered by pure culturing of microorganisms, however, it is known that less than 1% of all microorganisms can be obtained in pure cultures. The remaining majority of microorganisms is only viable by close biological interactions provided in microbial communities and is not available for enzyme discovery using the classical pure culture approaches. The investigation of microbial communities, which can be viewed as metaorganisms, has been enabled during the last two decades by refining established methods for the analysis of genes, mRNA or proteins and are called metagenomics, metatranscriptomics and metaproteomics, respectively. To date, these techniques have mostly been used in the field of microbial ecology for the understanding of the composition, function and metabolism of microbial communities but not for the purpose of bioprospecting for novel enzymes. Identification of genes that code for possible enzyme candidates is hindered, due to the fact that 30-40% of the sequenced metagenomes contain genes coding for unidentified proteins. Additionally, the -omics techniques generate large amounts of data that need to be analyzed and the outcome of the analysis does not necessarily lead to the discovery of novel applicable enzymes.The work presented in this thesis describes the establishment of the necessary conditions for a metaproteomics-based method that allows a straightforward and targeted identification of novel enzymes with desired activity from microbial communities. The approach provides a valuable alternative to the incomplete and inefficient analysis of non-targeting data and laborious workflow, which is typically generated by the established meta-omics techniques. In developing the methods presented in this thesis, microbial communities in constructed environments were established, which allowed for the controlled expression of extracellular hydrolytic enzymes under defined conditions. By combination and modulation of advanced metaproteomics and metagenomics techniques, we were vi able to directly identify the enzymes and the corresponding gene sequences of several cellulolytic enzymes as a first exam...

show abstract

“…Unlike standard SDS-PAGE gels, the proteases are prepared for electrophoresis under non-reducing conditions (Vandooren et al, 2013). Therefore, the proteases can be kept biologically active.…”

mentioning

confidence: 99%

“…Therefore, the proteases can be kept biologically active. After electrophoresis, the proteases are separated out by size and a preceding renaturation step allows the proteases to cleave the co-polymerised substrate in the gel (Vandooren et al, 2013). This leaves a measureable band in the gel.…”

mentioning

confidence: 99%

Biological and chemical hazards in water-mix metalworking fluids and mists

Brookes¹

View full text Add to dashboard Cite

show abstract

Zymography methods for visualizing hydrolytic enzymes

Cited by 261 publications

References 72 publications

Cleistanthin A inhibits the invasion and metastasis of human melanoma cells by inhibiting the expression of matrix metallopeptidase‑2 and ‑9

Cleistanthin A inhibits the invasion and metastasis of human melanoma cells by inhibiting the expression of matrix metallopeptidase‑2 and ‑9

Methods development for metaproteomics-guided bioprospecting of novel enzymes

Biological and chemical hazards in water-mix metalworking fluids and mists

Contact Info

Product

Resources

About