Catalase and superoxide dismutase double staining zymogram technique for Deinococcus and Kocuria species exposed to multiple stresses

Abstract

Superoxide dismutase (SOD) and catalase expression is associated with oxidative stress. Existing techniques for the individual staining of SOD and catalase have been described in the past. The objective of this study was to achieve a simple and rapid technique for the double staining of bacterial SOD and catalase on the same polyacrylamide gel. SOD detection was carried out using nitro-blue tetrazolium (NBT) dye reduction followed by ferricyanide precipitation for negative staining of the catalase enzyme on the same gel. The staining procedure resulted in pale blue SOD bands while catalase appeared as yellow bands against a greenish blue background on the same gel. This technique was used to detect changes in the polymorphic forms of these enzymes in Deinococcus radiodurans R1 and Kocuria sp. C2 subjected to stresses like UV and gamma radiation and desiccation.

A role for antioxidants in acclimation of marine derived pathogenic fungus (NIOCC 1) to salt stress

“…Cell free extracts for extracellular, and the fungal cells grounded with PBS buffer for intracellular catalase subjected to different salt stress was resolved in 8% non denaturing PAGE in Tris-glycine buffer using the method of Shukla et al [38] with minor modification. Briefly, the resolved gel was washed twice with distilled water immediately and rinsed with 50 mM phosphate buffer (pH 7.8) containing 0.01 M H 2 O 2 .…”

Two-dimensional zymography in detection of proteolytic enzymes in wheat leaves

“…The most popular systems, arranged in a hierarchy of Google search results, have been elaborated for detection of: phosphatases (Robinson and Glew 1980), reactive oxygen species scavenging enzymes: catalases, superoxide dismutases, peroxidases (Shukla et al 2009;Srivalli and Khanna-Chopra 2001), amylases (Steup and Gerbling 1983) and proteinases (Lantz and Ciborowski 1994). Furthermore, the system has been successfully used for detection of glycohydrolases: glucanases and glucosidases, xylanases, mannases, hemicellulases, cellulases and ligninases (Joo et al 2009;Royer and Nakas 1990;Sakamoto and Toyohara 2009) as well as esterases, lipases (Kwon et al 2011) and nucleases (Cazenave and Toulme 2001).…”

Modifying Polyacrylamide Background Color for the Nitroblue Tetrazolium-Based Superoxide Dismutase Staining Assay

“…Other variations include modest changes in the riboflavin concentration [12], inclusion of ethylenediaminetetraacetate (EDTA) [11] (which promotes superoxide generation via the light-dependent reduction of free flavins and by chelating trace metals that inhibit superoxide generation [13] [14]), and a simplified methodology that combines NBT and riboflavin application together as a single solution [11]. A significant research obstacle in the development of these assays is the extensive fine-tuning of reagents required to achieve effective band-background contrast and/or to visualize multiple enzymes (e.g., See reference [10]). A straightforward technique for changing the background color for enzyme visualization would therefore be advantageous to assay developers.…”

“…There have since been published multiple variations of this assay in response to specific experimental requirements. For example, some variations employ a lower concentration of NBT [10] [11] this study] because the original concentration prescribed [9] would be prohibitively expensive for routine analysis and with larger staining solutions. Another variation of the NBT-based assay includes an assay for the simultaneous detection of catalase on the same gel [10].…”

“…For example, some variations employ a lower concentration of NBT [10] [11] this study] because the original concentration prescribed [9] would be prohibitively expensive for routine analysis and with larger staining solutions. Another variation of the NBT-based assay includes an assay for the simultaneous detection of catalase on the same gel [10]. Other variations include modest changes in the riboflavin concentration [12], inclusion of ethylenediaminetetraacetate (EDTA) [11] (which promotes superoxide generation via the light-dependent reduction of free flavins and by chelating trace metals that inhibit superoxide generation [13] [14]), and a simplified methodology that combines NBT and riboflavin application together as a single solution [11].…”