A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.
The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.
Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.
Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.
1Department of Neurobiology and Anatomy, University of Utah School of Medicine, 2Department of Molecular, Cellular and Developmental Biology, UCSB, 3Howard Hughes Medical Institute, 4Stowers Institute for Medical Research
An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.
1Department of Biochemistry and Molecular Medicine, University of California, Davis, 2NSF Center for Biophotonics Science & Technology, University of California, Davis, 3University of Tromsø, 4Department of Surgery (Division of Surgical Oncology), University of California, Davis, 5UC Davis Comprehensive Cancer Center, University of California, Davis, 6Department of Biological Chemistry, University of California, Davis
Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.
1Department of Pathology, New York University Langone School of Medicine, 2Program in Molecular Pathogenesis, Marty and Helen Kimmel Center for Biology and Medicine and Skirball Institute for Biomolecular Medicine, 3Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 4Veteran Affairs New York Harbor Healthcare System
This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.
1Research Service, Veterans Affairs Greater Los Angeles Healthcare System, 2Department of Urology, The David Geffen School of Medicine, University of California, Los Angeles, 3GeneFluidics, 4Division of Infectious Diseases, Veterans Affairs Greater Los Angeles Healthcare System, 5Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles
We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.
Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.
This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.
This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.
Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.
1Department of Electrical and Computer Engineering, Boston University, 2Department of Biomedical Engineering, Boston University, 3Center for Advanced Genomics Technology, Boston University, 4Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, 5Department of Microbiology, Boston University School of Medicine, 6CNR (National Research Council), Istituto di Chimica del Riconoscimento Molecolare
Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.
Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.
1Dynamique des Interactions Hôte Pathogène, Institut Pasteur, Paris, France, 2Imagopole, Institut Pasteur, Paris, France, 3Pathogenomique Mycobacterienne Integrée, Institut Pasteur, Paris, France
We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.
We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.
We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.
Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.
In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.
This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.
In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.
Tangential Flow Ultrafiltration: A “Green” Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles
Tangential flow ultrafiltration (TFU) is a recirculation method used for the weight-based separation of biosamples. TFU was adapted to size-select (1-20 nm diameter) and highly concentrate a large volume of polydisperse silver nanoparticles (4 L of 15.2 μg ml-1 down to 4 ml of 8,539.9 μg ml-1) with minimal aggregation.
1The Warren Alpert Medical School of Brown University, 2Laboratorio de Investigacion de Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, 3Department of International Health, Johns Hopkins Bloomberg School of Public Health, 4Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College London
The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.