An excellent guide on the analysis of proteins on SDS-PAGE gels, through staining with coomassie blue dye and western blot analysis. Analysis of Protein Gels (SDS-PAGE). David R. Caprette, Rice University. - [Read Analysis of Protein Gels (SDS-PAGE)]
This protocol includes an ammonium sulfate cut, affigel blue chromatography and affinity chromatography. Mike A. Dyer Lab Harvard. - [Read Antibody Purification]
This assay is used when working with phage vectors carrying the beta-gal gene. If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-gal gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta-gal will work as indicator host bacteria; a single chromosomal copy of the gene is not a problem. - [Read Assay for Phage Containing the Beta-galactosidase Gene]
This assay is used when working with phage vectors carrying the beta-galactosidase gene (often used for immunological screening). If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-galactosidase gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta- galactosidase will work as indicator host bacteria. - [Read Assay for Phage Containing the Beta-galactosidase Gene Protocol]
BN-PAGE has become the method of choice for the investigation of the respiratory protein complexes of the electron transfer chains of a range of organisms. It allows the separation in two dimensions of extremely hydrophobic protein sets for analysis and also provides information on their native interactions. In this review we discuss the capabilities of BN-PAGE in proteomics and the wider investigation of protein:protein interactions with a focus on its use and potential in plant science. - [Read Blue-Native PAGE in Plants: A Tool in Analysis of Protein-Protein Interactions]
The bradford dye-binding assay is a colorimetric assay for measuring total protein concentration. It involves the binding of Coomassie Brilliant blue to protein. There is no interference from cations nor from carbohydrates such as sucrose.
Detergents such as sodium dodecyl sulfate and triton x-100 can interfere with the assay, as well as strongly alkaline solutions.
Includes a general overview of the procedure and preparation of the standards in the protocol. - [Read Bradford Assay Method]
cDNA Library Screening Protocol - ZAPII Detailed Protocol. XLI-Blue MRF' and cDNA phage library screening. Dr. Laurie. University of Virginia Health Science Center - [Read cDNA Library Screening Protocol - ZAPII]
The potential cytotoxicity of compounds under hypoxic conditions is determined by exposing cell cultures to test compounds in a low oxygen atmosphere. Subsequent cell survival is determined by the MTT and methylene blue colorimetric assays. - [Read Colorimetric Cytotoxcity Assays for Anchorage Dependent Cells Protocol]
Protocol for combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. Includes: Cell preparations and BrdU labeling; Detection of antigen by immunocytochemistry (ICC); Visualization of ICC antigen; -Gal-BCIG reaction (for producing a blue precipitate visible under brightfield microscopy); Cell processing for in situ hybridization; In situ hybridization (ISH); etc... - [Read Combined DNA In Situ Hybridization and Immunocytochemistry Protocol]
Coomassie Blue Staining Protocol- http://cgap-mf.nih.gov/Protocols/DNARNAProteomicAnalysis/Proteomics/StainingProcedures.html#Coomassie
Skin fibroblasts are incorporated into 3-D collagen lattices containing the test compounds. An inhibition of lattice contraction indicates a possible toxic effect which is verified by trypan blue exclusion for cell viability. - [Read Human Skin Fibroblast/Collagen Lattice Cytotoxicity Test]
Lowry Protein Assay. The Lowry procedure is one of the most venerable and widely-used protein assays, being first described in 1951 [Lowry et al., J. Biol. Chem. 193: 265-275 (1951)]. Under alkaline conditions, copper complexes with protein. When folin phenol reagent (phospho-molybdic-phosphotungstic reagent) is added, the Folin-phenol reagent binds to the protein. Bound reagent is slowly reduced and changes color from yellow to blue. P.J. Hansen, Dept. of Animal Sciences, University of Florida. - [Read Lowry Protein Assay]
The cytotoxic effect of chemicals upon mammalian cells, such as BALB/c 3T3 and HepG2, in culture is measured by highest tolerated dose (HTD), cell viability (Neutral Red) and total cell protein (coomassie blue). - [Read Neutral Red Cytotoxicity Assay Protocol]
Protocol describes oviduct transfer of mouse embryos. It is based on the Whittingham method, which applied a well described procedure for the rat to the mouse. It is best to practice this procedure first on a cadaver and then on an anesthetized 0.5-dpc pseudopregnant mouse using a dye solution or blue Affigel beads rather than embryos to gain experience in finding the opening of the oviduct (infundibulum). - [Read Oviduct Transfer Protocol]
Triazine dyes, such as Cibacron Blue 3GA, can be linked to a hexyl spacer arm and then immobilized on a polyhydroxyl support matrix that has been activated with either 1,1-carbonyldiimidazole or epichlorohydrin. An alternative procedure for immobilizing dyes using the direct coupling method is provided in Immobilization of Dyes on Polyhydroxyl Matrices Using the Direct Coupling Method. - [Read Preparation of Affinity-Ligand Resins by Immobilization of Dyes on Polyhydroxyl Matrices]
Protein Staining Protocols- http://cgap-mf.nih.gov/Protocols/DNARNAProteomicAnalysis/Proteomics/StainingProcedures.html
Protocols for protein staining. Includes: Coomassie Blue Staining; Silver Nitrate Staining; Zinc Imidazole Negative Staining. - [Read Protein Staining Protocols]
Methods on protein trafficking. Information on N5 Secretion and protein trafficking. Methods include: Alcian Blue Test; Lucifer Yellow uptake assay; Preparation of total protein extracts for western immunoblots; Screen for Drug Sensitivity. - [Read Protein Trafficking Methods]
Colloidal coomassie blue staining, Destaining, Reduction/Alkylation, Peptide extraction For MALDI-MS analysis and For MALDI-MS analysis protocols and methods. Institut Curie Paris. - [Read Proteomic protocols and Peptide Extraction]
A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.
Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.
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.
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 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.