applied Protocols

Category: DNA Protocols: EMSA DNA-Protein Protocols

A non-radioactive electrophoretic mobility shift assay based on the digoxigenin detection system had been developed and applied to the analysis of the interaction between the heat shock transcription factor and the heat shock element of N. crassa. (Bioch - [Read A non-radioactive electrophoretic mobility shift assay for the detection of heat shock element (HSE)]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses a single thermostable RNA polymerase to perform high-specificity RT-PCR.  A high-temperature RT reaction is followed by PCR amplification of the cDNA using a single thermostable poymerase, the GeneAmp AccRT RNA PCR enzyme from Applied Biosystems.  The high temperature of the RT reaction enhances the specificity of primer binding and also reduces secondary structure in the template, thereby increasing the efficiency of polymerization. - [Read Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium]

Category: Cell Biology and Cell Culture: Apoptosis Protocols

Apoptosis, Cell Death, And Proliferation. Roche Manual. Includes a complete list of apoptosis methods. Roche. - [Read Apoptosis, Cell Death, And Proliferation. Roche Manual.]

Category: Cell Biology and Cell Culture

Protocol applies EFs to cells in vitro but has been modified and to use electrotactic chambers to accommodate cells growing in planar culture or in three-dimensional (3D) gels, en bloc tissue cultures in 3D and possible small embryos, such as that from frog and zebra fish. The EF is applied to the cells or tissues cultured in a customer designed electrotactic chamber via agar salt bridges, Steinberg’s solution and Ag/AgCl electrodes. - [Read Application of Direct Current Electric Fields to Cells and Tissues in vitro]

Category: Cytogenetics Protocols: Spectral Karyotyping SKY Protocols

SKY has been applied to various tumor groups including hematological malignancies, sarcomas, carcinomas and brain tumors, with the intent of identifying specific chromosomal abnormalities that may provide insight to the genes involved in the disease process as well as identifying recurrent cytogenetic markers for clinical diagnosis and prognostic assessment. - [Read Applications of SKY in Cancer Cytogenetics]

Category: Genetics and Genomics: Cancer Genetics Protocols

SKY has also been applied for the mouse genome, enabling investigators to extrapolate information from mouse models of cancer to their human counterparts. This review will address the advances that SKY has facilitated in the field of cancer cytogenetics, as well as its variety of application in the cancer research laboratories. - [Read Applications of SKY in Cancer Cytogenetics Review]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

This protocol describes how to use DIG Chem-Link to directly label any DNA [e.g. plasmids, PCR products, cDNA prepared from mRNA] or RNA (e.g. total RNA, poly(A)+ mMRNA). The DIG Chem-Link or Biotin Chem-Link may also be used to label oligonucleotides. Includes: Required Purity of DIG Chem-Link Templates; Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Key Product Required for Direct Labeling of DNA or RNA; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol for Chem-Link labeling of DNA or RNA with DIG or Biotin. Includes: Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Choosing the right labeling method for your hybridization experiment. Includes: Homogeneous labeling methods for DNA; Homogeneous labeling methods for RNA; Stability of probe-target interaction; Nonradioactive labeling of oligonucleotides; Double-stranded versus single-stranded probes. - [Read Choosing the Right Labeling Method for your Hybridization Experiment]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. Includes: Cell preparations and BrdU labeling; Detection of antigen by immunocytochemistry (ICC); Visualization of ICC antigen; -Gal-BCIG reaction (for producing a blue precipitate visible under brightfield microscopy); Cell processing for in situ hybridization; In situ hybridization (ISH); etc... - [Read Combined DNA In Situ Hybridization and Immunocytochemistry Protocol]

Category: Protein Protocols: Protein Quantitation Concentration Protocols

A Comparision of Different Protein Determination Methods and protocols in table format. The Wolfson Centre for Applied Structural Biology The Hebrew University of Jerusalem Dr. Mario Lebendiker. - [Read Comparison Different Protein Quantitation Determination Methods]

Category: Reporter Gene Assays: GFP Assay Protocols

The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells. - [Read Constructing and Expressing GFP Fusion Proteins]

Category: Microscopy Protocols: Optical Microscopy Protocols

The visibility of the faint star light is enormously enhanced against a dark background. This principle is applied in darkfield (also called darkground) microscopy, a simple and popular method for making unstained transparent specimens clearly visible. Such objects often have refractive indices very close in value to that of their surroundings and are difficult to image in conventional brightfield microscopy. - [Read Darkfield Illumination]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Three Ambion kits were used to quantitate specific miRNAs and to detect differential miRNA expression in various mouse brain regions and cell types isolated by laser capture microdissection (LCM). These techniques can be applied to studying miRNA in other species, tissues, and cell types. Includes: Obtain Laser Capture Microdissected Samples; Isolate miRNA from LCM Samples; Quantitate miRNA by qRT-PCR. - [Read Detect and Quantitate MicroRNA in Laser Capture Microdissection Samples]

Category: Immunology

Protocol for detection of autoantibodies with self-assembling radiolabeled antigen tetramers. Details how to produce radiolabeled antigen-streptavidin tetramers for detection of antibodies by immunoprecipitation. Optionally, the antigen tetramers can be denatured to compare responses to folded and unfolded antigen in the same system. This technique can be applied to a large or small number of samples, and a given sample can be simultaneously assayed with multiple antigens. - [Read Detection of Autoantibodies with Self-Assembling Radiolabeled Antigen Tetramers Protocol]

Category: Cytogenetics Protocols

CGH provides a comprehensive picture of all chromosomal gains and losses within a single experiment. In contrast to banding analysis, CGH requires no preparation of metaphase chromosomes from the cells to be analyzed (which in certain tumor types may be difficult or even impossible). - [Read Detection of Chromosomal Imbalances Using DOP-PCR and Comparative Genomic Hybridization (CGH)]

Category: Cytogenetics Protocols: Spectral Karyotyping SKY Protocols

Protocol for the Detection of chromosomal imbalances using DOP-PCR and comparative genomic hybridization (CGH). Includes: Preparation and labeling of DOP-PCR products; Comparative genomic hybridization (CGH). - [Read Detection of Chromosomal Imbalances using DOP-PCR and Comparative Genomic Hybridization (CGH)]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol for detection of even-skipped transcripts in Drosophila embryos with PCR/DIG-labeled DNA probes. - [Read Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of even-skipped transcripts in drosophila embryos with PCR/DIG-labeled DNA probes. This protocol has been used to detect the transcript distribution of a number of genes by in situ hybridization, including evenskipped and seven-up, in whole mount Drosophila embryos, and engrailed Antennapedia in whole mount grasshopper embryos. Includes: Probe labeling; Evaluation of labeling reaction; Preparation of embryos, hybridization and detection.
 - [Read Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of HPV 11 DNA in paraffin-embedded laryngeal tissue with a DIG-labeled DNA probe. Includes: Probe preparation; Pretreatment of slides; Preparation of tissue sections; Hybridization; Washes; Detection of hybrids. - [Read Detection of HPV 11 DNA in Paraffin-Embedded Laryngeal Tissue with a DIG-Labeled DNA Probe Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probes. - [Read Detection of mRNA in Tissue Sections Using DIG-labeled RNA and Oligonucleotide Probes Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNA on paraffin embedded material of the central nervous system with DIG-labeled RNA probes. - [Read Detection of mRNA on Paraffin Embedded Material of the Central Nervous System with DIG-Labeled RNA]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNAs on cryosections of the cardiovascular system using DIG-labeled RNA probes. Protocol was optimized from a protocol using 35S-labeled RNA probes. It allows to detect the expression of low abundant mRNAs in the cardiovascular system, e.g. of the proinflammatory cytokine GM-CSF in normal human coronary arteries, and of IL6 and gp130 in human failing hearts. The protocol can be combined with immunohistochemistry. - [Read Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol describes here a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. Includes: In situ hybridization with DIG-labeled probes; Detection of DIG-labeled probes; Fluorescence microscopy. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol describes a high sensitivity indirect detection procedure for DIG-labeled hybridization probes.  The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization.  This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes. 
    - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol]