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Direct Elisa Using Fluorescent Substrate Protocol - http://www.abcam.com/assets/pdf/protocols/Direct%20ELISA%20using%20fluorescent%20substrate%20protocol.pdf

Category: ELISA Protocols

Direct Elisa Using Fluorescent Substrate Protocol. Abcam. - [Read Direct Elisa Using Fluorescent Substrate Protocol]

Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4483

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

MEF feeders are prepared weekly to provide a substrate for undifferentiated embryonic stem (ES) cells. Primary MEF cells are thawed, established in culture, treated with mitomycin C to halt their proliferation so they cannot overgrow the ES cultures, and then replated onto dishes convenient for ES cell culture. This protocol can also be used to prepare feeder cells from STO fibroblast cell lines. - [Read Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates Protocol]

Alkaline Phosphatase (APAAP) Technique Protocol - http://www.nottingham.ac.uk/pathology/protocols/apap.html

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

Protocol for alkaline phosphatase (APAAP) technique. Includes: Fixation; Technique; Substrate Solution. - [Read Alkaline Phosphatase (APAAP) Technique Protocol]

Growing Adherent Cells on Coverslips or Multiwell Slides Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4289

Category: Cell Culture Protocols

Glass is an excellent substrate for most tissue-culture-adapted cells and is compatible with all fixing and staining solutions. Glass coverslips in tissue-culture dishes or in 24-well multiwell plates are suitable carriers, as are multiwell slides. For high-resolution studies, choose glass coverslips of the highest available grade; #1 or #1.5 coverslips are the appropriate thickness. - [Read Growing Adherent Cells on Coverslips or Multiwell Slides Protocol]

CD90 (Thy-1) Antibody Staining Protocol for Immunohistochemistry - http://www.ihcworld.com/_protocols/antibody_protocols/cd90_pharmingen.htm

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

Protocol for CD90 (Thy-1) antibody staining for Immunohistochemistry. Includes: Primary Antibody; Antigen Retrieval; Detection Methods; Chromogen Substrate; Counterstain. - [Read CD90 (Thy-1) Antibody Staining Protocol for Immunohistochemistry]

Attaching Suspension Cells to Slides Using a Cytocentrifuge Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4291

Category: Cell Culture Protocols: Cell Staining Protocols

A simple method for handling suspension cells is to attach them to a solid substrate before fixation, and one way to do this is to use a cytocentrifuge. - [Read Attaching Suspension Cells to Slides Using a Cytocentrifuge Protocol]

Synchronization of Mammalian Cell Cultures in Mitosis Using Selective Detachment Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4489

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: Mitosis Protocols

Protocol describes a method for synchronizing monolayer cells in mitosis using selective detachment from their substrate. During mitosis, cells become spherical, causing them to become more loosely attached to their substrate. The "rounded up" cells are selectively detached by tapping the culture flask, resulting in a population in which as many as 90-98% of the cells are in mitosis. The drug nocodazole is used to increase the percentage of cells undergoing mitosis before detachment is performed - [Read Synchronization of Mammalian Cell Cultures in Mitosis Using Selective Detachment Protocol]

A Method for Assaying Deubiquitinating Enzymes - http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=140113

Category: Protein Protocols: Protein Ubiquitination Protocols

A Method for Assaying Deubiquitinating Enzymes. Lee et al., Biol Proced Online. May 1998. A general method for the assay of deubiquitinating enzymes was described in detail using 125I-labeled ubiquitin-fused Î±NH-MHISPPEPESEEEEEHYC (referred to as Ub-PESTc) as a substrate. - [Read A Method for Assaying Deubiquitinating Enzymes]

Protein Kinase C Assay - http://cancer.ucsd.edu/newtonlab/Newton%20Web%20PKC%20ASSAY.html

Category: Signalling Signal Transduction Protocols: Protein Kinase Protocols

Protein Kinase C Assay, Newton lab. Using the peptide substrate Ac-FKKSFKL-NH2. Detailed Assay Protocol. - [Read Protein Kinase C Assay]

Quantitative GUS Activity Assay in Intact Plant Tissue Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4688

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

The activity of ß-glucuronidase (GUS) can be accurately determined in intact plant tissue using 4-methylumbelliferyl ß-D-glucuronide (4-MUG) as a substrate. Upon hydrolysis by GUS, the fluorochrome 4-methyl umbelliferone (4-MU) is produced. This method is based on the permeability of both 4-MUG and 4-MU through plant tissue. It consists of incubation of the tissue with the reagent and quantification of the fluorescence emitted by 4-MU in the solution. GUS activity in each sample can be... - [Read Quantitative GUS Activity Assay in Intact Plant Tissue Protocol]

Alkaline Phosphatase (APAAP) Technique - http://www.nottingham.ac.uk/pathology/protocols/apap.html

Category: Signalling Signal Transduction Protocols

Technique for Alkaline Phosphatase (APAAP). Techniques includes information on: Cytological Preparations, Fixation, Technique, Results, Substrate Solution and 0.005M Tris/HC1 Saline Buffer pH 7.6. - [Read Alkaline Phosphatase (APAAP) Technique]

ELISA Method to Measure Inhibition of the COX Enzymes Protocol - http://www.natureprotocols.com/2006/11/22/an_elisa_method_to_measure_inh.php

Category: ELISA Protocols

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. - [Read ELISA Method to Measure Inhibition of the COX Enzymes Protocol]

Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in 96-Well Plates - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4160

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

Method described indicates how cells are lysed by a simple freeze/thaw protocol and treated with a chemiluminescent substrate. - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in 96-Well Plates]

Assay of ß-Galactosidase in Yeast: Permeabilized Cell Assay - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4158

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

In the method described here, the cells are permeabilized to allow the substrate to enter the cells and the activity is normalized to the number of cells assayed. - [Read Assay of ß-Galactosidase in Yeast: Permeabilized Cell Assay]

Synchronization of Mammalian Cell Cultures in Mitosis Using Selective Detachment Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4489

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

This protocol describes a method for synchronizing monolayer cells in mitosis using selective detachment from their substrate. During mitosis, cells become more spherical, causing them to become more loosely attached to their substrate. The "rounded up" cells are selectively detached by tapping the culture flask, resulting in a population in which as many as 90-98% of the cells are in mitosis. The drug nocodazole is used to increase the percentage of cells undergoing mitosis before detachment.. - [Read Synchronization of Mammalian Cell Cultures in Mitosis Using Selective Detachment Protocol]

VEGF Antibody Staining Protocol - http://www.ihcworld.com/_protocols/antibody_protocols/vegf_vg1_chemicon.htm

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

Protocol for VEGF antibody staining. Includes: Primary Antibody; Antigen Retrieval; Detection Methods; Chromogen Substrate; Counterstain. - [Read VEGF Antibody Staining Protocol]

Histone H1 Kinase Activity Assay - http://www.bio.com/protocolstools/protocol.jhtml?id=p910

Category: Signalling Signal Transduction Protocols

This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography. Includes information: H1 Kinase Assay on Individual Xenopus Oocytes; H1 Kinase Assay on Xenopus Egg Extract Samples; H1 Kinase Assay on Tissue Culture Cells; Helpful protocol hints. - [Read Histone H1 Kinase Activity Assay]

Western Analysis Using the Chemiluminescent Alkaline Phosphatase Substrate CSPD Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4256

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Use of the chemiluminescence-producing alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD, also known as adamantyl-1,2-dioxetane phosphate), or its dioxetane relatives provides a substantial increase in sensitivity over colorimetric substrates and radiochemical methods currently used for the detection of antigen-antibody complexes immobilized on nylon or PVDF membranes. - [Read Western Analysis Using the Chemiluminescent Alkaline Phosphatase Substrate CSPD Protocol]

Quantitative GUS Activity Assay of Plant Extracts - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4690

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

Using excitation at 365 nm and measuring emission at 455 nm, the amount of 4-MU produced can be quantified. Under these conditions, background fluorescence from the substrate is negligible, especially if the appropriate filter is selected. - [Read Quantitative GUS Activity Assay of Plant Extracts]

Direct Retrieval of DNA Fragments from Pulsed-field Gels Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4083

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for direct retrieval of DNA fragments from pulsed-field gels. A gel slice containing a fragment of DNA resolved by pulsed-field gel electrophoresis is treated with agarase. The released DNA can be used as a substrate for ligation or restriction without further purification. - [Read Direct Retrieval of DNA Fragments from Pulsed-field Gels Protocol]

Polycrystalline thin films Sample Preparation Protocol MALDI - http://prowl.rockefeller.edu/methods/thinfilm.htm

Category: Mass Spectrometry Protocols

This method of sample preparation produces a uniform layer of very small crystals on the mass spectrometer's sample stage that are mechanically well adhered to the substrate. It is a variant of the dried droplet method. PROWL - [Read Polycrystalline thin films Sample Preparation Protocol MALDI]

Antibody Addition to Drosophila Specimens and Detection Using Enzyme-Linked Reagents Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4526

Category: Drosophila Protocols

Enzyme-linked reagents give excellent sensitivity and use a simple light microscope for detection. A range of enzymes is available, but for staining in situ, horseradish peroxidase will suit most needs. Diaminobenzidine (DAB) is one of the most sensitive substrates for horseradish peroxidase. It yields an intense brown product that is insoluble in both water and alcohol. It can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. - [Read Antibody Addition to Drosophila Specimens and Detection Using Enzyme-Linked Reagents Protocol]

FAK Autophosphorylation Assay - http://www.bio.com/protocolstools/protocol.jhtml?id=p1984

Category: Signalling Signal Transduction Protocols

This kinase assay is meant to determine whether an agonist or event can influence the autophosphorylation of FAK. The addition of 1 Î¼l of polyGT to the kinase reaction mix will determine the activity of the enzyme against a substrate. Includes information on: Harvest, Immunoprecipitation, Kinase Reaction and Antibody Detection of FAK. - [Read FAK Autophosphorylation Assay]

Working with Bacteriophage P1 and Its Cloning Systems Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4003

Category: Viral Manipulation Protocols: Phage Protocols

Protocol describes methods for recovery and purification of recombinant clones of bacteriophage P1 or PAC DNAs from bacteria. Because of their large size, these DNAs are sensitive to shearing forces and must be handled carefully. This protocol generally yields P1 DNA that works well as a substrate or template in enzymatic reactions. - [Read Working with Bacteriophage P1 and Its Cloning Systems Protocol]

Making a scanning array: in situ synthesis of oligonucleotides on a solid substrate - http://www.oup.co.uk/pdf/pas/6-5-3.pdf

Category: DNA Microarray Protocols

Making a scanning array: in situ synthesis of oligonucleotides on a solid substrate. Elder et al., Oxford Practical Approach Series. - [Read Making a scanning array: in situ synthesis of oligonucleotides on a solid substrate]

Articles (1-4 of 4)

Genomic DNA Labeling of Microarrays Protocol

DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array.

Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

This Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays.

Immobilized Antigen Phage Antibody Selection Protocol

A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.

Histone H1 Kinase Activity Assay

Histone H1 Kinase Activity Assay Protocol. This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography.

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