enzymatic Protocols

Category: DNA Protocols: DNA Extraction Protocols: Extracting Ancient DNA

The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, but PCR can be used to amplify and study short mitochondrial DNA sequences thatare of anthropological and evolutionary significance. SVANTE PAABO, PNAS. - [Read Ancient DNA: Extraction, characterization, molecular cloning, and enzymatic amplification. PDF]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: TUNEL assay apoptosis

Apoptosis Detection Using Terminal Transferase and Biotin-16-dUTP (TUNEL -Enzyme Method). IHC World - [Read Apoptosis Detection Using TUNEL Enzymatic]

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: PCR Protocols

Protocol for dealing with carryover contamination in PCR- enzymatic strategy. Repeated use of PCR and manipulation of its products cause aerosols that can contaminate neighboring samples and work areas.  Such "carryover contamination" can be prevented by including dUTP in place of dTTP for all amplification reactions. - [Read Dealing with Carryover Contamination in PCR: An Enzymatic Strategy Protocol]

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: 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]

Category: PCR Protocols: PCR Optimization

Method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.  The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. - [Read Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization Protocol]

Category: Immunology: Immunocytochemistry Protocols

Staining Tissue, Staining Cells Smeared on Slides. Positive staining controls, Negative staining controls, Immunoenzymatic Technique and FAQ. R & D Systems. - [Read Enzymatic Protocol for Immunocytochemistry]

Category: Immunohistochemistry Protocols

Protocol for immunohistochemistry on fixed, paraffin-embedded sections. This method is widely used and applies to the detection of the overwhelming majority of antigens, with few exceptions for which enzymatic retrieval is required. The method uses a strong chelating agent, EDTA. Includes: Double indirect AP; AP Developing solution; Indirect immunohistochemistry with avidin-biotin and HRP; HRP Developing solution. - [Read Immunohistochemistry on Fixed, Paraffin-Embedded Sections Protocol]

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: Protein Protocols: Protein Extraction From Cells

Preparation of cell lysates from yeast by enzymatic digestion of the cell wall. - [Read Preparation of cell lysates from yeast by enzymatic digestion of the cell wall]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for the purification of DNA recovered from agarose and polyacrylamide gels by anion-exchange chromatography. Fragments of DNA recovered from agarose gels are sometimes poor templates or substrates in subsequent enzymatic reactions.  This problem can be solved by binding the DNA to a positively charged matrix, such as DEAE-Sephadex or DEAE-Sephacel, in buffers of low ionic strength.  After washing the matrix, the DNA is eluted by raising the strength of the buffer. - [Read Purification of DNA Recovered Anion-exchange Chromatography Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol for rat Chromaffin cells primary cultures: Standardization and quality assessment for single-cell assays. Includes: Preliminars; Isolation of the rat adrenal medulla; Enzymatic digestion of medullary tissue; Collecting and culturing the cells. - [Read Rat Chromaffin Cells Primary Cultures Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

A fragment of gel containing a band of DNA is excised and digested with agarase, which hydrolyzes the polymer to disaccharide subunits.  The released DNA is then purified by phenol extraction and ethanol precipitation.  The method works well for DNAs ranging in size from <5 kb to >20 kb. - [Read Recovery of DNA from Low-melting-temperature Agarose Gels: Enzymatic Digestion with Agarase Protocol]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol describes a split luciferase complementation assay used to study the interaction of proteins in cells. In the split protein strategy, a single reporter protein/enzyme (firefly luciferase [Fluc]) is cleaved into amino-terminal and carboxy-terminal halves; each half is fused to one of two interacting proteins, X & Y. Physical interactions between the two proteins reconstitute the functional reporter protein, leading to enzymatic activities that can be measured by in vitro or in vivo assay - [Read Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Cells]

Category: Immunohistochemistry Protocols

Protocol provides the sensitivity of enzymatic detection (HRP) in immunohistochemical procedures with the versatility and convenience of fluorescence detection. - [Read Tyramide Amplification and Synthesis Protocol]

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]

Category: Cell Culture Protocols

Includes information on: Cell Isolation Theory ( Tissue Types: Epithelial Tissue, Connective Tissue); Dissociating Enzymes (Collagenase, Trypsin, Elastase, Hyaluronidase, Papain, Chymotrypsin, Deoxyribonuclease I, Neutral Protease (Dispase), Trypsin Inhibitor (Soybean)); Cell Isolation Techniques: Working With Enzymes, Basic Primary Cell Isolation, Equilibration with 95%O2:5%CO2, Trituration, Enzymatic Cell Harvesting, Cell Adhesion and Harvesting, Trypsin for Cell Harvesting, etc... - [Read Worthington Tissue Dissociation Guide]