Protein Microarray Chips - An Introduction. Introduction, Types of Protein Chips, Attachment, Protein and Antibody Chip Production, Applications of Protein Chips, Detection methods, and Future Directions.
Molecularstation. - [Read Protein and Antibody Microarray Chips - An Introduction]
Western Blot Home- http://www.molecularstation.com/protein/western-blot/
Western Blot Home. Information on western blotting, western blot procedure and methods, western blot books, stripping protocols. - [Read Western Blot Home]
Covers Many Western Blot Problems and Includes Many Solutions. Fuzzy Bands, Low or Weak Signal, High Background, Spots on Film, Too Many Bands. A MolecularStation Guide. - [Read Troubleshooting Western Blot]
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]
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]
Protocol describes how to prepare double-stranded RNA (dsRNA) for RNA interference in Drosophila by synthesis of individual RNA strands from linearized plasmid templates, followed by annealing of the strands. DsRNA molecules with a length of 500-800 bp seem to be most active. The dsRNA can be made from cDNA or genomic DNA templates, as long as most of the dsRNA corresponds to presumptive exon sequence. - [Read Synthesis of dsRNA for RNAi in Drosophila: Plasmid Template Method Protocol]
Protocol describes the preparation of subtracted cDNA probes by hybridization to an mRNA driver, followed by purification of the single-stranded radiolabeled cDNA by hydroxyapatite chromatography. Before preparing the probe, it is a good idea to have filters (which contain the cDNA library to be screened) ready to hybridize. - [Read Synthesis of Radiolabeled, Subtracted cDNA Probes Using Oligo(dT) as a Primer Protocol]
A synthetic oligonucleotide annealed to single-stranded DNA derived from a recombinant bacteriophage M13 or phagemid template is used to prime the synthesis of complementary radiolabeled DNA. Synthesis is catalyzed by the Klenow fragment of E. coli DNA polymerase I, which extends the annealed primer for various distances along the single-stranded template DNA. - [Read Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates Protocol]
Technique yields a heterogeneous population of short radiolabeled molecules 200-300 nucleotides in length. These probes are synthesized, as in Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates, by extension of an oligonucleotide primer on a single-stranded DNA template. The radiolabeled products of the reaction are then separated from the template by electrophoresis through a denaturing gel from which they are eluted directly into hybridization buffer. - [Read Synthesis of Single-stranded DNA Probes of Heterogeneous Length from Bacteriophage M13 Templates]
T Cell Depletion by Cytotoxic Elimination Protocol- https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E66365BA79D5D5A9DA4948DDF019E3B&objectid=6674981906D4B89C730A2E1ED1987295
Protocol describes removal of T cell subsets by cytotoxic elimination using CD-specific antibodies. Describes the complete removal of T cells from lymphocyte preparations based on the presence of the glycoprotein Thy-1 on the cell surface of T lymphocytes. Cytotoxic elimination is employed; however, Thy-1-specific antibodies are used rather than MHC class II-specific antibodies so that T cells are eliminated rather than B cells and accessory cells. - [Read T Cell Depletion by Cytotoxic Elimination Protocol]
Describes T cell enrichment using cytotoxic antibodies, and also describes the depletion of T cells and their subpopulations using the same approach. In the latter unit, T cell surface markers (Thy-1, CD4, and CD8) are targeted by the cytotoxic antibodies. - [Read T Cell Enrichment by Cytotoxic Elimination of B Cells and Accessory Cells Protocol]
T Cell Enrichment By Nonadherence to Nylon Protocol- https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E66363CC65D4418DAE176108087071E&objectid=667493A4BF337BA420CD91E90B60CA95
Protocol describes a convenient, although imprecise, means of enriching T cells through removal of accessory and B cells; use of nylon wool is preferred if both of the latter subsets are to be removed, while Sephadex is used when the goal is primarily to remove accessory cells. - [Read T Cell Enrichment By Nonadherence to Nylon Protocol]
TAIL PCR Protocol. TAIL is a series of reactions that are intended to map where a T-DNA (transfer DNA) has inserted within the genome. The main components of the 3 reactions are the AD (Arbitrary Degenerate) primers, border primers, and DNA from the T-DNA - [Read TAIL PCR Protocol]
TAIL PCR Protocol. TAIL is a series of reactions that are intended to map where a T-DNA (transfer DNA) has inserted within the genome. The main components of the 3 reactions are the AD (Arbitrary Degenerate) primers, border primers, and DNA from the T-DNA - [Read TAIL PCR Protocol]
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.
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.
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.
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.