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Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-density Gradient

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Detection of D-glutamate Production from the Dual Function Enzyme, 4-amino-4-deoxychorismate Lyase/D-amino Acid Transaminase, in Mycobacterium smegmatis

Authors: Helen K. Opel-Reading, Roman Mortuza and Kurt L. Krause, date: 01/05/2019, view: 1345, Q&A: 0

[Abstract] D-amino acid transaminase (D-AAT) is able to synthesize both D-glutamate and D-alanine, according to the following reaction: D-alanine + α-ketoglutarate ⇌ D-glutamate + pyruvate. These two D-amino acids are essential components of the peptidoglycan layer of bacteria. In our recently published work, MSMEG_5795 from Mycobacterium smegmatis ...

Extraction and Analysis of Bacterial Teichoic Acids

Authors: Kelvin Kho and Timothy C. Meredith, date: 11/05/2018, view: 1962, Q&A: 0

[Abstract] Teichoic acids (TA) are anionic polymers comprised of polyol phosphate repeat units that are abundant in the cell wall of Gram-positive bacteria. Both wall teichoic acid (WTA) and lipoteichoic acid (LTA) play important roles in regulating cell wall remodeling as well as conferring antibiotic resistance. To analyze TA, we describe a polyacrylamide ...

Extraction and Quantification of Polyphosphate (polyP) from Gram-negative Bacteria

Authors: Jan-Ulrik Dahl, Lihan Xie and Ursula Jakob, date: 09/20/2018, view: 1790, Q&A: 0

[Abstract] Polyphosphate (polyP), a universally conserved biomolecule, is composed of up to 1,000 phosphate monomers linked via phosphoanhydride bonds. Reaching levels in bacteria that are in the high nmoles per mg protein range, polyP plays important roles in biofilm formation and colonization, general stress protection and virulence. Various protocols for ...

Determination of Polyhydroxybutyrate (PHB) Content in Ralstonia eutropha Using Gas Chromatography and Nile Red Staining

Authors: Janina R. Juengert, Stephanie Bresan and Dieter Jendrossek, date: 03/05/2018, view: 5044, Q&A: 0

[Abstract] Ralstonia eutropha H16 produces and mobilizes (re-utilizes) intracellular polyhydroxybutyrate (PHB) granules during growth. This protocol describes the visualization of intracellular Nile red stained PHB granules and the quantification of PHB by gas chromatography. Our first method describes how to analyze PHB granules by fluorescence ...

Identification and Quantification of Secondary Metabolites by LC-MS from Plant-associated Pseudomonas aurantiaca and Pseudomonas chlororaphis

Authors: Izzah Shahid, Muhammad Rizwan and Samina Mehnaz, date: 01/20/2018, view: 4376, Q&A: 0

[Abstract] Increased antibiotic resistance of plants and human pathogens and continuous use of chemical fertilizers has pushed microbiologists to explore new microbial sources as potential antagonists. In this study, eight strains of Pseudomonas aurantiaca and Pseudomonas chlororaphis, have been isolated from different plant sources and ...

Determination of Mn Concentrations in Synechocystis sp. PCC6803 Using ICP-MS

Authors: Fabian Brandenburg, Hanan Schoffman, Nir Keren and Marion Eisenhut, date: 12/05/2017, view: 3300, Q&A: 0

[Abstract] Manganese (Mn) is an essential micronutrient for all photoautotrophic organisms. Two distinct pools of Mn have been identified in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), with 80% of the Mn residing in the periplasm and 20% in cytoplasm and thylakoid lumen (Keren et al., 2002). In this protocol, we ...

Quantification of Bacterial Polyhydroxybutyrate Content by Flow Cytometry

Authors: Antonio Lagares (Jr.) and Claudio Valverde, date: 12/05/2017, view: 3071, Q&A: 0

[Abstract] We describe here a detailed protocol for the quantification of the intracellular content of polyhydroxybutyrate (PHB) in a population of bacterial cells by flow cytometry, which is based on a consensus of several previously reported works.

Detection of Reactive Oxygen Species (ROS) in Cyanobacteria Using the Oxidant-sensing Probe 2’,7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA)

Authors: Rajneesh, Jainendra Pathak, Ananya Chatterjee, Shailendra P. Singh and Rajeshwar P. Sinha, date: 09/05/2017, view: 8535, Q&A: 0

[Abstract] Reactive oxygen species (ROS) are cell signaling molecules synthesized inside the cells as a response to routine metabolic processes. In stress conditions such as ultraviolet radiation (UVR), ROS concentration increases several folds in the cells that become toxic for the cell survival. Here we present the method for in vivo detection of ...

Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS

Authors: Marina Borisova and Christoph Mayer, date: 08/05/2017, view: 4169, Q&A: 0

[Abstract] We describe here in detail a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based method to determine N-acetylmuramic acid-6-phosphate (MurNAc-6P) in bacterial cell extracts. The method can be applied to both Gram-negative and Gram-positive bacteria, and as an example we use Escherichia coli cells in this ...

Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803

Authors: Yvonne Zilliges and Ramon Damrow, date: 07/20/2017, view: 4135, Q&A: 0

[Abstract] Cyanobacteria synthesize a variety of chemically-different, high-value biopolymers such as glycogen (polyglucose), poly-β-hydroxybutyrate (PHB), cyanophycin (polyamide of arginine and aspartic acid) and volutin (polyphosphate) under excess conditions. Especially under unbalanced C to N ratios, glycogen and in some cyanobacterial genera ...

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