Protocols in Current Issue
    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: 786, 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 ...
    Flow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages
    Authors:  Evgeniya V. Nazarova, Maria Podinovskaia, David G. Russell and Brian C. VanderVen, date: 02/20/2018, view: 3086, Q&A: 0
    [Abstract] Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb ...
    Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis
    Authors:  Liku B Tezera, Magdalena K Bielecka and Paul T Elkington, date: 07/20/2017, view: 4142, Q&A: 0
    [Abstract] Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the ...
    Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry
    Authors:  Manish Bhuwan, Madhuri Suragani, Nasreen Z. Ehtesham and Seyed E. Hasnain, date: 01/20/2017, view: 5844, Q&A: 0
    [Abstract] The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway ...
    Separation of Free and Bound cAMP in Mycobacteria
    Authors:  Arka Banerjee and Sandhya S. Visweswariah, date: 07/20/2016, view: 4205, Q&A: 0
    [Abstract] Mycobacterial genomes encode a plethora of genes that are involved in the synthesis, utilization and degradation of cAMP. The genome of M. tuberculosis H37Rv, for example, encodes 16 adenylyl cyclases and 10 genes harbouring the cyclic nucleotide-binding (CNB) domain (Shenoy and Visweswariah, 2006). Cyclic AMP is efficiently secreted by ...
    Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni2+-NTA Plate
    Authors:  Allexa Dow and Sladjana Prisic, date: 03/05/2016, view: 5100, Q&A: 0
    [Abstract] The displacement assay was designed to quantify the direct competition between two homologous ribosomal proteins from Mycobacterium tuberculosis, S18-1 and S18-2, for interaction with their cognate binding partner, ribosomal protein S6 (Prisic et al., 2015). The S18 proteins were dialyzed in two physiologically relevant ...
    Isolation of Genomic DNA from Mycobacterium Species
    Authors:  Pawan Kumar, Soumitra Marathe and Sangeeta Bhaskar, date: 03/05/2016, view: 8188, Q&A: 1
    [Abstract] Isolation of genomic DNA from Mycobacterium species has been a tedious procedure. This can primarily be attributed to thick and waxy cell wall of mycobacteria which hampers lysis of the bacterial cell. We have tested various approaches to isolate mycobacterial DNA and based on this, an optimized protocol is presented here. This protocol ...
    Extraction and Purification of Mycobacterial Mycolic Acids
    Authors:  Christian M. Dupont and Laurent Kremer, date: 10/20/2014, view: 8377, Q&A: 0
    [Abstract] Mycolic acids are major long-chain fatty acids, containing up to 80-90 carbon atoms that represent essential components of the mycobacterial cell wall (Pawelczyk and Kremer, 2014). Each mycobacterial species possesses a specific mycolic acid profile characterized by various chemical modifications that decorate the lipid. Mycolic acids play a ...
    Determination of Rifampicin-resistance Mutation Frequency and Analysis of Mutation Spectra in Mycobacteria
    Authors:  Liang-Dong Lyu and Guo-Ping Zhao, date: 07/05/2014, view: 7378, Q&A: 0
    [Abstract] Understanding the genetic safeguarding mechanism of Mycobacterium tuberculosis (Mtb) may help us to explain i), how Mtb survive the genetic assaults elicited by both reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host macrophages and ii), why some strains of Mtb, e.g., Mtb strains from East Asian ...
    Analysis of Mycobacterial Protein Secretion
    Authors:  Alka Mehra and Jennifer A. Philips, date: 06/20/2014, view: 8638, Q&A: 0
    [Abstract] Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Analysis of proteins secreted by Mtb has been of interest to the field of tuberculosis research since certain secreted proteins interact with the host to promote virulence, while others may be important antigens or serve as biomarkers of infection. Here, we describe ...

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