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Restriction enzyme digestion of DNA: basic method - http://www.methodbook.net/dna/restrdig.html

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

How much DNA to digest? Master mix for restriction digest. Double digests. Star activity. Matt Lewis, Department of Pathology, Univ. Liverpool. - [Read Restriction enzyme digestion of DNA: basic method]

Gel Mobility Shift Assay in PDF format - http://www.biochem.northwestern.edu/ibis/morimoto/Protocols/III.%20Proteins/C.%20DNA-Protein%20Interactions/3a.%20GMSA.pdf

Category: DNA Protocols: EMSA DNA-Protein Protocols

This protocol works well for HSF DNA-binding activity experiments. - [Read Gel Mobility Shift Assay in PDF format]

Factors that Influence Restriction Enzyme Digestion Activity - http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/cuteffects.html

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]

Analysis of protein phosphorylation: methods and strategies PDF - http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1365-313X.2005.02613.x

Category: Protein Protocols: Protein Phosphorylation Protocols

Paper describing methods for monitoring kinase activity, investigating kinaseâ€“substrate specificity, examining phosphorylation in planta and the determination of phosphorylation sites in a protein. In addition, strategic considerations for experimental design and variables will be discussed. Scott C. Peck, the Plant Journal. - [Read Analysis of protein phosphorylation: methods and strategies PDF]

Protein Purification: Assays, Specific Activity, Initial Fractionation - http://wine1.sb.fsu.edu/bch5425/lect29/lect29.htm

Category: Protein Protocols: Protein Purification and Identification

Protein Purification: Assays, Specific Activity, Initial Fractionation. Blaber Lecture. - [Read Protein Purification: Assays, Specific Activity, Initial Fractionation]

Chemotaxis Assay - http://www.cbrinstitute.org/labs/springer/protocols/melissa_chemotaxis.html

Category: Immunology: Chemotaxis

Chemotaxis Assay, Springer Lab. A chemotaxis assay's function is to assess whether a factor or molecule of interest has chemotactic activity on a motile cell type. Chemotaxis is the ability of a factor to cause the migration of a cell. The chemotactic assay is based on the creation of a chemical gradient of the chemotactic agent which will cause cells to migrate through the gradient towards the chemotactic agent. - [Read Chemotaxis Assay]

Analysis of Mitochondrial Membrane Potential with the Sensitive Fluorescent Probe JC-1 - http://www.cyto.purdue.edu/flowcyt/research/cytotech/apopto/data/cossar1/cossariz.htm

Category: Cell Organelle Protocols: Mitochondria Protocols

The technique of JC-1 staining has been developed with the intent to detect DY in intact, viable cells. For this purpose JC-1 acts as a marker of mitochondrial activity, since the formation of J-aggregates, which give red emission, is reversible. Cells with high DY are those forming J-aggregates, thus showing high red fluorescence. On the other hand, cells with low DY are those in which JC-1 maintains (or re-acquire) monomeric form, thus showing only green fluorescence. - [Read Analysis of Mitochondrial Membrane Potential with the Sensitive Fluorescent Probe JC-1]

Immunoprecipitation / Kinase Assay - http://www.upstate.com/misc/protocols.q.prot.e.ipkinaseassay/Immunoprecipitation+%2F+Kinase+Assay

Category: Signalling Signal Transduction Protocols: Protein Kinase Protocols

Immunoprecipitation / Kinase Assay Protocol. Upstate. Protocol applicable only to kinases whose activity is not altered by cell lysis or immunoprecipitation procedures, and do not require soluble cofactors for activity. - [Read Immunoprecipitation / Kinase Assay]

Photosynthesis and Respiration - Introduction - http://homepages.gac.edu/~cellab/chpts/chpt8/intro8.html

Category: Plant Biology Protocols: Photosynthesis and Respiration Protocols

The physiological reactions of mitochondria and chloroplasts can be reduced to a series of electron transfers, catalyzed by specific enzymes found within the organelles. Thus, we can study the component processes of photosynthesis and respiration by isolating the organelles and measuring specific enzyme activity associated with that organelle. - [Read Photosynthesis and Respiration - Introduction]

Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4157

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

Method describes how a crude extract is prepared, and the activity is normalized to the amount of protein assayed. This method is particularly suitable for comparing cells that are grown under very different conditions or that have different genetic backgrounds. - [Read Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts]

In-Gel Kinase Assay - http://www.bio.com/protocolstools/protocol.jhtml?id=p911

Category: Signalling Signal Transduction Protocols

Protocol describes how to assay for kinase activity within a polyacrylamide gel, rather than in solution. The advantages to an in-gel assay are that an apparent molecular weight can be assigned to the kinase activity and that multiple kinase activities can be distinguished. Includes protocol hints. - [Read In-Gel Kinase Assay]

Assay of β-Galactosidase in Yeast Protocol - http://www.oardc.ohio-state.edu/stockingerlab/Protocols/BetaGalAssays.pdf

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

There are two basic methods for the in vitro assay of B-galactosidase activity from yeast. They differ mainly in the method of preparing the material for assay. Both methods are described with accompanying protocols. Method I: Assay of Crude Extracts includes: Yeast Cell Growth; Yeast Cell Harvest; B-gal assays; Bradford Assays. Method II: Permeabilized cell assay. - [Read Assay of β-Galactosidase in Yeast Protocol]

(PI 3-Kinase) Activity Assay - http://www.upstate.com/misc/protocol_detail.q.prot.e.assay.a.name.e.Phosphatidyl_Inositol-3_Kinase_Assay_%28PI3%20Kinase%20Assay%29

Category: Signalling Signal Transduction Protocols

Protocol describing (PI 3-Kinase) activity assay. Includes Preparation of Phosphatidylinositol (PI). - [Read (PI 3-Kinase) Activity Assay]

Passage of Adherent Cell Lines (subculture). - http://www.hyclone.com/pdf/procedure_passage.pdf

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Remove medium from culture container, rinse with PBS, expose cells to trypsin, incubate, stop trypsin activity by adding serum containing medium, dissociate cells, dilute and return to incubator. - [Read Passage of Adherent Cell Lines (subculture).]

Immunocytochemistry in Free-Floating Sections Protocol - http://www.fisher.co.uk/techzone/life/table-pdfs/protocol/immunocytochemistry_netwells_protocol.pdf

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]

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]

Assay for Immunoprecipitated Phosphoinositide 3-Kinase (PI 3 Kinase) Activity (in vitro) - http://www.upstate.com/misc/protocols.q.prot.e.assay/Assay+for+Immunoprecipitated+Phosphoinositide+3+Kinase++PI+3+Kinase++Activity+%3Ci%3E+in+vitro+%3C%2Fi%3E

Category: Signalling Signal Transduction Protocols: Protein Kinase Protocols

Immunoprecipitation assay for Phosphoinositide 3-Kinase (PI 3 Kinase) Activity (in vitro) Protocol. Upstate. - [Read Assay for Immunoprecipitated Phosphoinositide 3-Kinase (PI 3 Kinase) Activity (in vitro)]

Induction and Measurement of Cytotoxic T Lymphocyte Activity Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E663458DAAA03E5A23DC69D6BE8BE3E&objectid=6674B15EE255310CC4C2307B31AE3411

Category: Immunology: T Cell Protocols

Describes generating CTL against some commonly used target antigens. Two methods for the quantitation of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions. - [Read Induction and Measurement of Cytotoxic T Lymphocyte Activity Protocol]

3-7 Assay Protocol - http://www.promega.com/tbs/tb323/tb323.pdf

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Protocol for 3/7 assay. Includes: Detection of Caspase-3 and -7 Activities in Cell-Based Assays; Detection of Caspase-3 or -7 Activity Using Purified Caspases. - [Read 3-7 Assay Protocol]

Chemotaxis Assay Protocol - http://www.cbrinstitute.org/labs/springer/protocols/melissa_chemotaxis.html

Category: Cell Biology and Cell Culture

This chemotaxis assay protocol is based on the premise of creating a gradient of the chemotactic agent and allowing cells to migrate through a membrane towards the chemotactic agent. A chemotaxis assay can determine whether your protein or small molecule of interest has chemotactic activity on a specific cell type. Chemotaxis is then the ability of a protein to direct the migration of a specific cell. - [Read Chemotaxis Assay Protocol]

In Vitro MAP Kinase Assay - http://www.whitelabs.org/Lab%20Protocols/kinase%20and%20phosphatase%20assays/In%20Vitro%20MAP%20kinase%20assay%20%209.htm

Category: Signalling Signal Transduction Protocols: MAPK Protocols

Protocol describes an in vitro assay to detect MAP kinase activity using PC12 cells. - [Read In Vitro MAP Kinase Assay]

Mapping Protein-binding Sites on DNA by Dnase I Footprinting Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3947

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Dnase I is used to fragment a radiolabeled target DNA in the presence and absence of a nuclear extract. A "footprint" is generated when a protein binds to the target and protects a specific segment of DNA from the nucleolytic activity of Dnase I. By comparing the electrophoretic mobility of the Dnase I cleavage products to those of a sequence ladder derived from the same DNA fragment, the position(s) of the DNA sequences recognized by DNA-binding proteins can be determined. - [Read Mapping Protein-binding Sites on DNA by Dnase I Footprinting Protocol]

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: 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]

A Sensitive ELISA-Based Assay for Glycosyltransferases - http://www.glycotech.com/protocols/Proto4.html

Category: Molecular Biology Protocols: Carbohydrate Protocols

The method allows the detection and quantification of glycosyltransferase activity using an ELISA-based procedure and carbohydrate-specific monoclonal antibodies. Avoids the use of radiolabeled substrates. Bruce A. Macher~Professor of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA - [Read A Sensitive ELISA-Based Assay for Glycosyltransferases]

Articles (1-4 of 4)

In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

DNA Ligation Protocol

The DNA Ligation protocol described here contains the steps required to join together using ligase enzyme both plasmid DNA and insert DNA fragments in order to create a new plasmid. This new ligated plasmid can be transformed after into competent bacteria to produce DNA for mini, midi or maxi-prep isolation.

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol. This protocol contains the steps for 3' end rapid amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly-A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.

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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