secondary Protocols

Category: Cloning Protocols: Baculovirus Protein Expression Protocols

Protocol for amplification of Baculovirus plaques and preparation of high titer virus stocks. Includes: Primary Amplification; Secondary Amplification. - [Read Amplification of Baculovirus Plaques and Preparation of High Titer Virus Stocks Protocol]

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: RNA Protocols: cDNA Libraries Library

Includes procedures for cDNA screens using secondary and tertiary screening. Dr. Bart Frank Lab. - [Read Benton Davis Blots for cDNA Libraries]

Category: Antibody Protocols: Antibody Labeling Protocols

Once tissues are fixed and permeabilized, the antibodies are added. These antibodies can be labeled directly or detected by a labeled secondary reagent. For indirect detection, any reagent that binds specifically to the primary antibody can be "tagged" and used to locate the antibody. The possible reagents include anti-immunoglobulin antibodies, protein A or G, or, if the first antibody is labeled with biotin, streptavidin. They can be labeled with enzymes or gold. - [Read Binding Antibodies to Tissue Sections Protocol]

Category: Immunohistochemistry Protocols: Blocking Protocols

Protocols for blocking solutions. Includes: Endogenous peroxidase blocking solution (H2O2-Azide); Non-specific antibody background blocking solution: swine serum; Non-specific antibody background blocking solution: MILK; Enzymatic antigen retrieval solution; Unconjugated, biotin- and fluorochrome-conjugated antibody (primary) solutions; Unconjugated, biotin- and fluorochrome-conjugated antibody (secondary) solutions; Enzyme-conjugated secondary antibody solutions.; etc.. - [Read Blocking Solutions Protocols]

Category: Molecular Imaging: Immunofluorescence Microscopy

Cell Staining for Immunofluorescence Microscopy. Includes protocols for fixing the cells, Coverslip Preparation for Adherent Cells, Coverslip Preparation for Non-Adherent Cells, Paraformaldehyde Fixation, and Methanol/Acetone Fixation. Blocking protocols include blocking with primary antibody, and incubation with secondary antibody. - [Read Cell Staining for Immunofluorescence Microscopy]

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Because sequencing reactions catalyzed by thermostable DNA polymerases such as Taq are carried out at elevated temperatures, problems caused by mismatched annealing of primers or templates rich in secondary structure are greatly alleviated. - [Read Dideoxy-mediated Sequencing of DNA Using Taq DNA Polymerase Protocol]

Category: Cytogenetics Protocols: Spectral Karyotyping SKY Protocols

Protocol for DOP-PCR Secondary (SKY). - [Read DOP-PCR Secondary SKY Protocol]

Category: Immunology: Immunoassays

Protocol for ELISA assay for NGF. Includes: ABSORPTION OF THE POLYCLONAL AND PREIMMUNE SERUM; BLOCKING; SAMPLE PREPARATION; PREPARATION OF NGF STANDARDS; PROTEIN RECOVERY; DESIGNING THE PLATE; APPLYING THE STANDARDS AND SAMPLES; APPLYING THE MONOCLONAL; APPLYING SECONDARY ANTIBODY; APPLYING STREPTAVIDIN; CHROMAGEN DEVELOPMENT; READING THE PLATE. - [Read Enzyme-Linked ImmunoSorbent Assay (ELISA) for NGF Protocol]

Category: Antibody Protocols: Epitope Mapping Antibody Protocols

A set of overlapping synthetic peptides is synthesized, each corresponding to a small segment of the linear sequence of a protein antigen and arrayed on a solid phase. The panel of solid-phase peptides is then probed with a test antibody, and bound antibody is detected using an enzyme-labeled secondary antibody. This method is very rapid and can be extraordinarily successful. - [Read Epitope Mapping Using Synthetic Biotin-Labeled Peptides Protocol]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Protein Protein Interactions Protocols

This protocol describes the first step in constructing an array: amplification of the predicted ORFs that are to be included in the array. Gene-specific primers containing vector-specific flanking sequences that facilitate recombinational cloning are used to amplify each ORF. A secondary amplification can be used to extend the length of the homologous vector sequence flanking the ORF. - [Read Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Amplification of ORFs]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol describes the direct detection of RNA on DNA microarrays using Hybrid Capture (HC) technology and the HC ExpressArray Kit developed by Diagene.  The kit uses a proprietary antibody that binds specifically to RNA:DNA hybrids and a second, fluorescently labeled, antibody that detects the primary antibody.  Total RNA is applied directly to a glass-spotted DNA microarray, and stable RNA:DNA hybrids are visualized via a Cy3-labeled secondary antibody. - [Read Hybridization and Detection Using the HC ExpressArray Kit Protocol]

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

Protocol for Immunoblot. Includes: Staining and Laser Capture Microdissection; Protein Separation by Polyacrylamide Gel Electrophoresis; Electrophoretic Transfer To a Membrane (Nylon, PVDF or Nitrocellulose); Primary and Secondary Antibody Incubations; Visualization. - [Read Immunoblot Protocol]

Category: Immunology: Immunofluorescence Protocols

Protocol for immunofluorescence labeling of cells. Includes: Cell Preparation; Fixation; Application of Primary Antibody; Application of Secondary Antibody and Evaulation. - [Read Immunofluorescence Labeling of Cells Protocol]

Category: Immunology: Immunofluorescence Protocols: Immunofluorescence Staining Protocols

Provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to fluorophore. - [Read Immunofluorescence Staining Protocol]

Category: Immunohistochemistry Protocols

Protocol for immunohistochemistry with AP-Conjugated (NBT/BCIP). Protocol extensively blocks slides, further diluting the primary antibody, lengthening the incubation and washing time, using a simple AP-conjugated secondary at high dilution and use a slow long development with the most powerful IHC development, NBT/BCIP. Includes: Single AP stainiing and Double AP staining. - [Read Immunohistochemistry with AP-Conjugated (NBT/BCIP) Protocol]

Category: DNA Protocols: DNA Sequencing Protocols

The inclusion of formamide in sequencing gels eliminates secondary structure in the DNA during electrophoresis. Formamide gels are particularly useful and almost a necessity when sequencing DNA templates with a G/C content >55%. - [Read Preparation of Denaturing Polyacrylamide Gels Containing Formamide Protocol]

Category: Nucleic Acid Electrophoresis Protocols

Protocol for preparation of denaturing polyacrylamide gels containing formamide.The inclusion of formamide in sequencing gels eliminates secondary structure in the DNA during electrophoresis.  Formamide gels are particularly useful and almost a necessity when sequencing DNA templates with a G/C content >55%. - [Read Preparation of Denaturing Polyacrylamide Gels Containing Formamide Protocol]

Category: Immunology: Immunoprecipitation Protocols

Immunoprecopitation method, the protein from the cell or tissue homogenate is precipitated in an appropriate lysis buffer by means of an immune complex which includes the antigen (protein), primary antibody and Protein A-, G-, or L-agarose conjugate or a secondary antibody-agarose conjugate - [Read Protocol Immunoprecipitation]