Zymography, Casein Zymography, and Reverse Zymography: Activity Assays for Proteases and their Inhibitors

Oliver, Gary W.; Stetler‐Stevenson, William G.; Kleiner, David E.

doi:10.1007/978-3-642-59816-6_5

Cited by 14 publications

(11 citation statements)

References 15 publications

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“…The alkaline protease produced from the strain was analyzed on 12% SDS-polyacrylamide gel containing 0.1% casein as copolymerized substrate as described by Oliver et al [10]. The staining and distaining of the gel was performed by Coomassie brilliant blue in a single step based on method given by Leber and Balkwill [11].…”

Section: Detection Of Protease Activity By Zymographymentioning

confidence: 99%

“…The activity of the partially purifi ed protease was measured at different pH values using the following buffers (0.5 M): Sodium phosphate (pH 7-7.5), Tris-HCl (8-9), sodium carbonate (pH [10][11][12]. Stability of the partially purifi ed enzyme at (pH 10 and 12) was studied by preincubating the enzyme in the respective buffers for 2, 4, 5 and 24 h and assaying the residual enzyme activity at optimum assay conditions.…”

Section: Characterization Of Alkaline Proteasementioning

confidence: 99%

“…In a report by Banik and Prakash [27] the endogenous protease activity in various commercially available laundry detergents of international companies was studied and the maximum protease activity was found at 50°C in pH range 10. [5][6][7][8][9][10][11].0 in all the tested laundry detergents.…”

Section: Detergent Stabilitymentioning

confidence: 99%

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Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated Bacillus species Y

Majumdar

Shivakumar

2010

Indian J Microbiol

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Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about 1.5 kb) of its 16S rDNA it was identifi ed as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purifi cation of the enzyme by ammonium sulfate (50-70% saturation) yielded 8-fold purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purifi ed enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C up to 2 h. The protease showed good hydrolysis specifi city with different substrates tested. The presence of Mn 2+ , Co 2+ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up to 3 h retaining almost 70-80% activity and 10-20% activity at room temperature (30°C) and 50°C, respectively, indicating the potential role of this enzyme for detergent application.

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Section: Detection Of Protease Activity By Zymographymentioning

confidence: 99%

Section: Characterization Of Alkaline Proteasementioning

confidence: 99%

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Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated Bacillus species Y

Majumdar

Shivakumar

2010

Indian J Microbiol

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“…This medium contains various compounds secreted by cells, including MMP 1, MMP 2, MMP 8, and MMP 9, and is recommended as the standard one (Oliver et al, 1999). The use of CM from the same sample allows variations caused by cell plating at dif ferent times to be avoided.…”

Section: Cellsmentioning

confidence: 99%

“…MMP proteolytic activity was determined by zymographic assay (Oliver et al, 1999). Casein (for MMP 1 and MMP 8 collagenases) and gelatin (for MMP 2 and MMP 9 gelatinases) were used as sub strates.…”

Section: Cellsmentioning

confidence: 99%

Matrix metalloproteinase activity in transformed cells exposed to an antioxidant

et al. 2015

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We showed that antioxidant N acetylcysteine (NAC, 2-10 mM) rapidly (in 2 h) and completely deactivated the activity of matrix metalloproteinases (MMPs) (MMP 2 and MMP 9 gelatinases and MMP 1 and MMP 8 collagenases) secreted by transformed 3T3 SV40 mouse fibroblasts into the medium. The same MMP inhibition took place in the cell free medium conditioned by HT 1080 fibroblasts. This suggests that the direct chemical interaction between NAC and MMP resulted in the loss of MMP activity. In addition to this inhibitory effect, NAC decreased MMP 1 and MMP 9 (but not MMP 2) production in the cell medium. However, the level of MMP 1 and MMP 9 inhibitors (TIMP 1) remained normal, indicating a shift in the balance between the enzyme and inhibitor. The correlation between MMP 2 and the tissue enzyme inhibitor TIMP 2 level was similar in control and NAC treated cells. Moreover, reorganization of collagen type I at the cell surface was observed. Taken together, our results suggest that NAC exposure results in extracellular matrix remodeling and a change in cellular functions.

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Assaying Proteases in Cellular Environments

Wang

2002

CP Protein Science

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To better study protease activation, regulation, and inhibition physiologically or pharmacologically, it is often advantageous to use cell-based systems. The introduction of the cellular environment poses additional challenges for the assay of the protease of interest. In this unit, various methods for monitoring protease activity in cell-based systems is discussed. The scope of the unit is restricted to mammalian cell system. It is also stressed that specific considerations or requirements for a particular protease or peptidase are often needed in order to optimize the assay.

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Zymography, Casein Zymography, and Reverse Zymography: Activity Assays for Proteases and their Inhibitors

Cited by 14 publications

References 15 publications

Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated Bacillus species Y

Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated Bacillus species Y

Matrix metalloproteinase activity in transformed cells exposed to an antioxidant

Assaying Proteases in Cellular Environments

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