enzyme Protocols

Category: ELISA Protocols

Qualitative ELISA (Enzyme-Linked Immunosorbent Assay) used for screening detection of anti-platelet antibodies or for detection of platelet-associated Ig (PAIg). Andrei Musaji Viral immunity and pathogenesis group, Universite Catholique de Louvain. - [Read ELISA for detection of platelet-associated Ig (PAIg)]

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

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.  This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. - [Read ELISA Method Measure Inhibition COX Enzymes]

Category: Neuroscience Protocols: Neuronal Stains Protocols

Protocol for enzyme histochemical stain. - [Read Enzyme Histochemical Stains Protocol]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Enzyme Immunoaffinity Chromatography—A Rapid Semi-quantitative Method. - [Read Enzyme Immunoaffinity Chromatography—A Rapid Semi-quantitative Method]

Category: Protein Protocols: Protein Precipitation Procols

Acetone Precipitation Protocol and Method. Nam Sun Wang. Univ. Maryland. - [Read Enzyme Purification by Acetone Precipitation]

Category: Protein Protocols: Protein Precipitation Procols

ENZYME PURIFICATION BY SALT (AMMONIUM SULFATE) PRECIPITATION. Prepared by Nam Sun Wang, Department of Chemical & Biomolecular Engineering. University of Maryland. - [Read Enzyme Purification by Salt Ammonium Sulfate Precipitation]

Category: Protein Protocols: Western Blot Protocol

ENZYME-ASSISTED IMMUNOELECTROBLOTTING (IEB OR WESTERN BLOTTING. E.P. Rybicki and Maud Purves. Department of Microbiology. Copper Staining of Gels, Indirect enzyme immunoassay, Staining Proteins on Membranes, Blotting Buffer, Staining of proteins in gels - [Read ENZYME-ASSISTED IMMUNOELECTROBLOTTING (IEB OR WESTERN BLOTTING)]

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: Molecular Biology Protocols: Restriction Enzyme Digestion

Factors that Influence Restriction Enzyme Digestion Activity. Buffer Composition, Incubation Temperature, DNA Methylation, Star Activity, Multiple Enzymes. Laura Austgen PhD et. al, Biomedical Hypertextbook, Colorado State Univ. - [Read Factors that Influence Restriction Enzyme Digestion Activity]

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 μl 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]

Category: Epigenetics and Methylation Protocols: Bisulfite Treatment Methylation Assay Protocol

A detailed protocol for bisulfite treatment of DNA. DNA is first digested with a restriction enzyme. DNA is denauted at 97C for 5min. Bisulfite protocol is included. Also DNA purification using the Promega Wizard DNA Cleanup kit in indicated. Fan Lab. UCL - [Read Fan Lab Bisulfite Treatment of Genomic DNA]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocols for gene expression and protein localization in Arabidopsis. Includes: Detection of the native protein; Detection of a recombinant version; Immunofluorescence detection in Arabidopsis protoplasts; Isolation of Arabidopsis seedling protoplasts; Subcellular localization of GUS-fusion proteins in Arabidopsis seedlings; Localization of Arabidopsis proteins with GUS in situ enzyme assay. - [Read Gene Expression and Protein Localization in Arabidopsis Protocols]

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

In this method, the nuclease BAL 31 is used to make uni- or bidirectional deletions in a segment of cloned DNA. BAL 31 is a complex enzyme and tends to digest a population of double-stranded DNA targets in an asynchronous fashion, Deletions created by BAL 31 are therefore far more heterogeneous in size than those created by processive enzymes such as exonuclease III. - [Read Generation of Bidirectional Sets of Deletion Mutants by Digestion with BAL 31 Nuclease Protocol]

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

The double-stranded DNA of recombinant plasmid, phagemid, or bacteriophage M13 replicative form DNA is digested with two restriction enzymes whose sites of cleavage both lie between one end of the target DNA and the binding site for universal primer. The enzyme that cleaves nearer the target sequence must generate either a blunt end or a recessed 3' terminus; the other enzyme must generate a four-nucleotide protruding 3' terminus. - [Read Generation of Sets of Nested Deletion Mutants with Exonuclease III Protocol]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast DNA Isolation Protocols

Protocol that isolates intact, high molecular weight DNA from yeast cells for subcloning and rare cutting restriction enzyme analysis. Using this protocol one can expect a yield of 100-200 µg of DNA per prep. - [Read High Molecular Weight Yeast Liquid DNA Preparation Protocol]

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

There are several strategies to visualize the antibody. For transmitted light microscopy, color development substrates for enzymes are often used. The antibody can be directly labeled with the enzyme. However, such a covalent link between an antibody and an enzyme might result in a loss of both enzyme and antibody activity. For these reasons several multistep staining procedures have been developed, where intermediate link antibodies are used. In this protocol use the Vectastain ABC-kit. - [Read Immunocytochemistry in Free-Floating Sections Protocol]

Category: Immunohistochemistry Protocols

Protocol for immunohistochemistry enzyme HRP staining. Includes: Cell Linee; Frozen Sections; Paraffin Sections; Pretreatments of Tissue Sections. - [Read Immunohistochemistry Enzyme HRP Staining Protocol]

Category: Immunohistochemistry Protocols

Protocol describes the application of peroxidase or alkaline phosphatase conjugates in the immunohistochemical labeling of formalin-fixed, paraffin-embedded tissue sections. Includes: Removal of Paraffin and Rehydration; Antigen Retrieval - Unmasking of Antigen; Enzyme retrieval; Microwave retrieval; Inactivation of Endogenous Peroxidase; etc.. - [Read Immunohistochemistry Protocol]

Category: PCR Protocols: Inverse PCR Protocols

Inverse PCR is used to amplify and clone unknown DNA that flanks one end of a known DNA sequence and for which no primers are available.  The technique involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region.  The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in the PCR. - [Read Inverse PCR Protocol II]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

Collagenase perfusion of rat liver yields a hepatocyte suspension which may be exposed to test compounds in order to assess their effects on cell viability and enzyme leakage. - [Read Isolation of Rat Hepatocytes Protocol]

Category: Plant Biology Protocols: Arabidopsis Tissue Culture

The most commonly used markers for selection of transgenic Arabidopsis are resistance to the antibiotic kanamycin and to the herbicide glufosinate ammonium. Resistance to kanamycin is conferred by a bacterial gene encoding the enzyme neomycin phosphotransferase (NPT). In this protocol, kanamycin-resistant seedlings are selected on solid medium. - [Read Kanamycin Selection of Transformed Arabidopsis Protocol]

Category: PCR Protocols

Protocol illustrates the rules of successful long PCR: No more than 1 ng of template DNA is used per microliter of PCR in a 100-µl reaction; approximately 0.1 µl of KlentaqLA (not plain Taq) is used per kilobase of target (for targets >10 kb, 1-1.3 µl of enzyme should be used); the Mg++ concentration is considered as the excess over the level of dNTPs. - [Read Long and Accurate PCR Protocol]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: Long PCR Protocols

General Guidelines for Long PCR Conditions and Enzyme Mixtures. Pete Estep. Harvard. - [Read Long PCR Reagents and Guidelines]

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]