signal Protocols

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Data Analysis Protocols

Provides several approaches to flow cytometry data analysis. Frequency determinations based on analysis of single-parameter fluorescence histograms and dual-parameter contour plots are presented. Steps are described for calculating values for signal-to-noise ratios when logarithmic amplification is used for data collection. - [Read Analysis of Flow Cytometry Data Protocol]

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols: Rat Neuronal Cell Culture Protocols

Conditional ablation of stat3/socs3 discloses the dual role for reactive astrocytes after spinal cord injury. The current protocol demonstrates that reactive astrocytes play a crucial role in wound healing and functional recovery by using mice with a selective deletion of the signal transducer and activator of transcription-3 (STAT3) or suppression of cytokine signaling-3 (SOCS3) under the control of Nestin gene promoter/enhancer (STAT3N–/–, SOCS3N–/–). - [Read Conditional Ablation of Stat3 Socs3 Discloses the Dual Role for Reactive Astrocytes]

Category: Transfection Protocols

Protocol should be viewed as a starting point for systematic optimization of transfection mediated by lipofecting agents. Once a positive signal has been obtained from a transfected plasmid carrying a standard reporter gene, optimal conditions for transfection can be established by systematic variation of parameters such as the initial cell density, the amount and purity of DNA, the media and serum, and the time of exposure of the cells to the cationic-lipid-DNA complex. - [Read DNA Transfection Mediated by Lipofection Protocol]

Category: Immunology: B Cell Protocols

B cell activation can be quantitated indirectly by assaying antibody production or directly by measuring cellular changes that occur immediately after exposure to an activation signal. Provides methods for the latter (direct) approach--namely, methods for quantifying early parameters of B cell activation such as increases in intracellular ionized calcium concentration [Ca2+]I, cell size, and MHC class II-antigen expression. - [Read Early Events in B Lymphocyte Activation Protocol]

Category: Drosophila Protocols: Drosophila Genetics Protocols

FISH protocols for Drosophila. Includes: RNA Probe Preparation; Embryo Collection and Fixation; Single FISH on Drosophila embryos; Post-Fixation, Hybridization and Post-Hybridization Washes; Development of FISH Signal; Storage, Mounting and Viewing of Samples; Double FISH on Drosophila Embryos; RNA-Protein Double Labeling; FISH on Dissected Tissues. - [Read FISH Protocols for Drosophila]

Category: Protein Protocols: Protein Phosphorylation Protocols

Fluorescent indicators for imaging protein phosphorylation in single living cells. Using Phocuses, genetically encoded fluorescent indicators, one can visualize signal transduction based on protein phosphorylation in living cells. Moritoshi Sato1, Takeaki Ozawa1, Kouichi Inukai2, Tomoichiro Asano2 & Yoshio Umezawa1. Nature Biotechnology - [Read Fluorescent indicators for imaging protein phosphorylation in single living cells]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

To reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. - [Read Immunoprecipitation: Preclearing the Lysate Protocol]

Category: Fungi Protocols: Neurospora Protocols

Procedure for an in vitro system to analyze light signal transduction in Neurospora crassa - [Read In Vitro System to Analyze Light Signal Transduction in Neurospora crassa]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Positron emission tomography (PET) is a established quantitative and noninvasive imaging modality. With the PET reporter gene (PRG)/PET reporter probe (PRP) system, based on a mutant form of herpes simplex virus 1 thymidine kinase (HSV1-sr39tk), the PET signal is directly proportional to the enzymatic activity of sr39TK9-14. In this protocol, we describe in detail a method for reporter gene labeling of islets and quantitative scanning using a reporter probe. - [Read In Vivo Functional Real-Time Imaging of Transplanted Islets Using Positron Emission Tomography (PET)]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

GFP serves as a molecular marker that can be imaged dynamically in living cells, both in its native form & as a fusion to other proteins. For GFP imaging, plants present the challenge of autofluorescence from chlorophyll, lignified cell walls, vacuolar contents, and other cell materials, all of which can obscure the GFP signal. Maximizing the signal-to-noise ratio is a major concern, and careful consideration should be given to the choice of tissue imaged, GFP expression level, etc. - [Read Live-Cell Imaging of GFP in Plants]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Optimized protocols for fluorescent in situ hybridization in Drosophila tissues. Includes: RNA Probe Preparation; Initial Embryo Fixation; Post-Fixation, Hybridization and post-Hybridization Washes; Development of FISH Signal; Mounting and Viewing of Samples; Double FISH; FISH on Dissected Tissues; RNA-Protein Double-labeling. - [Read Optimized Protocols for Fluorescent in situ Hybridization in Drosophila Tissues]

Category: Signalling Signal Transduction Protocols

Protocol demonstrates that reactive astrocytes play a crucial role in wound healing and functional recovery by using mice with a selective deletion of the signal transducer and activator of transcription-3 (STAT3) or suppression of cytokine signaling-3 (SOCS3) under the control of Nestin gene promoter/enhancer (STAT3N–/–, SOCS3N–/–). Procedure includes: Surgical procedures, Functional evaluation, Immunohistochemistry, In vitro migration assay. - [Read Protocol for Conditional Ablation of stat3/socs3 Discloses the Dual Role for Reactive Astrocytes]

Category: Signalling Signal Transduction Protocols: MAPK Protocols

This protocol uses mitogen-activated protein/ERK kinase (MEK) to activate the extracellular-signal-regulated (Erk) mitogen-activated protein (MAP) kinases upon agonist binding to receptors. Protocol includes information about: Harvest, Immunoprecipitation and Kinase Reaction. Also includes following solutions: Laemmli Sample Buffer (1X), ATP Mix, Kinase Buffer (10X), Lysis Buffer. Helpful protocol hints are also included. - [Read Raf-1 Kinase Assay]

Category: Proteomic Protocols: Silver Staining Protocols

Silver Staining Protocol. Compatible with mass spectrometry, excellent signal-to-noise ratio. Lammond Lab. - [Read Silver Staining Protocol]

Category: Protein Protocols: Western Blot Protocol: Troubleshooting Western Blots

Troubleshooting Guide for Chemiluminescent Western Blotting Pierce. High Background, Reverse image on film, Blot glows in the darkroom, Signal duration is less than 8 hours, Weak or no signal, Spots within the protein bands, non-specific bands. - [Read Troubleshooting Guide for Chemiluminescent Western Blotting Pierce]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for tyramide signal amplification (TSA) detection. - [Read Tyramide Signal Amplification (TSA) Detection Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for visualizing miRNAs in adult skeletal muscle by hybridization in situ. Includes: Visualizing miRNAs in adult skeletal muscle by hybridization in situ; Probe preparation; Tissue preparation; Fixation; Deproteinization with Proteinase K; Acetylation of the tissue; Dehydration; Hybridization step; Post-Hybridization Washes (Step 1-6 should be performed on shaker); Signal Detection (Steps should be performed on a shaker); Post-Detection Washes. - [Read Visualizing miRNAs in Adult Skeletal Muscle by Hybridization In Situ Protocol]

Category: Cytogenetics Protocols: RNA In Situ Hybridization Protocols

Protocol for visualizing miRNAs in adult skeletal muscle by hybridization in situ. Includes: Visualizing miRNAs in adult skeletal muscle by hybridization in situ; Probe preparation; Tissue preparation; Fixation; Deproteinization with Proteinase K; Acetylation of the tissue; Dehydration; Hybridization step; Post-Hybridization Washes (Step 1-6 should be performed on shaker); Signal Detection (Steps should be performed on a shaker); Post-Detection Washes. - [Read Visualizing miRNAs in Adult Skeletal Muscle by Hybridization in situ Protocol]

Category: Microscopy Protocols: Optical Microscopy Protocols

The light microscope allows dynamic biological processes to be imaged in their native (i.e., aqueous) environment with relatively high temporal resolution. However, the diffraction-limited resolution is low. When working at or beyond the diffraction-limited resolution of the LM, a disadvantage of fluorescence imaging is the relatively low signal-to-noise (S/N) ratio of the images. However, this can be increased significantly by video and computer technology. - [Read Watching Molecular Motors at Work by Video-Enhanced Light Microscopy]