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Troubleshooting Western Blot - http://www.molecularstation.com/protein/troubleshooting-western-blots/

Category: Protein Protocols: Western Blot Protocol: Troubleshooting Western Blots

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]

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High Efficiency LiAc Transformation Protocol - http://www.fhcrc.org/science/labs/gottschling/yeast/ytrans.html

Category: Yeast Protocols: Yeast Transformation Protocols: High Efficiency Yeast Transformation Protocols

Protocol for high efficiency LiAc transformation. - [Read High Efficiency LiAc Transformation Protocol]

High Efficiency Transformation Protocol - http://www.umanitoba.ca/faculties/medicine/biochem/gietz/method.html

Category: Yeast Protocols: Yeast Transformation Protocols: High Efficiency Yeast Transformation Protocols

Protocol for high efficiency transformation. - [Read High Efficiency Transformation Protocol]

High Iron Diamine Stain for Sulphomucins Protocol - http://www.nottingham.ac.uk/pathology/protocols/hidiam.html

Category: Neuroscience Protocols: Histology Stains Protocols: Mucin Stains Protocols

Protocol stain for sulphomucins- high iron diamine stain. - [Read High Iron Diamine Stain for Sulphomucins Protocol]

High Molecular Weight Yeast Liquid DNA Preparation Protocol - http://humgen.wustl.edu/hdk_lab_manual/yeast/yeast4.html

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]

High Throughput and Sensitive Assay Measure Yeast Cell Growth Protocol - http://www.natureprotocols.com/2006/11/22/highthroughput_and_sensitive_a.php

Category: Pharmacology and Toxicology Protocols: Drug Discovery

High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents. Method is highly sensitive, provides quantifiable data and offers high-throughput screening capability. Starting with the treatment of cells with different doses of damaging agents, pre-prepared growing media containing 96-well plates are inoculated and cell population is automatically monitored every 10 min for 48 hours. - [Read High Throughput and Sensitive Assay Measure Yeast Cell Growth Protocol]

High Throughput Western Blotting Protocol for Antibody Quality Control - http://discover.nci.nih.gov/abminer/files/Western.pdf

Category: Western Blot Protocols

Protocol for high throughput western blotting for antibody quality control. Includes: SDS-PAGE; TRANSFER; IMMUNOSTAINING; DETECTION. - [Read High Throughput Western Blotting Protocol for Antibody Quality Control]

High Yield Protein Production from Pichia pastoris Yeast PDF - http://www.nbsc.com/files/papers/abl_pichia.pdf

Category: Protein Protocols: Protein Expression Protocols

High Yield Protein Production from Pichia pastoris Yeast PDF. Julia Cino, PhD - [Read High Yield Protein Production from Pichia pastoris Yeast PDF]

High-Efficiency LiOAc Transformation of S. cerevisiae Protocol - http://www.biochem.emory.edu/labs/acorbe2/yeasttransformation.html

Category: Yeast Protocols: Yeast Transformation Protocols: High Efficiency Yeast Transformation Protocols

Protocol for high-efficiency LiOAc transformation of S. cerevisiae. - [Read High-Efficiency LiOAc Transformation of S. cerevisiae Protocol]

High-Efficiency Transformation of Yeast Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4145

Category: Yeast Protocols: Yeast Transformation Protocols: High Efficiency Yeast Transformation Protocols

Protocol for high-efficiency transformation of Yeast. - [Read High-Efficiency Transformation of Yeast Protocol]

High-resolution SNP mapping by denaturing HPLC - http://www.pnas.org/cgi/content/full/99/16/10575

Category: DNA Protocols: SNP Protocols

High-resolution SNP mapping by denaturing HPLC. A SNP mapping procedure that relies on resolving polymorphisms by denaturing HPLC without the necessity of determining the nature of the SNPs. They demonstrate the use of denaturing high-performance liquid chromatography to identify mutations in the candidate genes and to fine-map chromosomal breakpoints. - [Read High-resolution SNP mapping by denaturing HPLC]

High-Throughput Immunostaining Protocol - http://openwetware.org/wiki/Silver:_High-throughput_immunostaining

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

Protocol for high-throughput immunostaining. Includes: [edit] Detection of 14-3-3 in U2OS and HeLa cells by immunofluorescence. - [Read High-Throughput Immunostaining Protocol]

Home-made Taq Polymerase Purification - http://www.nottingham.ac.uk/~mbzspd/methods/Home_made_Taq_polyme.html

Category: PCR Protocols

PCR polymerase costs can be high. If you are willing to work, you can produce bacteria containing the clone. It appears to produce lots of Taq and is quite stable. The proceedure takes 4 days start to (15 000 units of Taq) finish. The Taq also appears ver - [Read Home-made Taq Polymerase Purification]

HPLC Protocol - http://tesla.ccrc.uga.edu/group_pages/protocols/downloads/HPLCProtocol.doc

Category: HPLC Protocols

HPLC Protocol. A general HPLC procedure to get you started on High Performance Liquid Chromatography. - [Read HPLC Protocol]

Hybridization of Oligonucleotide Probes in Aqueous Solutions Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3883

Category: Nucleic Acid Modification Protocols: Nucleic Acid Labeling Protocols

Hybridization is carried out in conventional aqueous solvents at a temperature well below the predicted melting temperature. Nonspecific hybrids are then removed by washing at high stringency in buffers containing quaternary salts. Tetramethylammonium chloride (TMACl) is used with probes that are 14-50 nucleotides in length, whereas tetraethylammonium chloride (TEACl) is used with longer oligonucleotides. - [Read Hybridization of Oligonucleotide Probes in Aqueous Solutions Protocol]

Image Based Assay of Endothelial Cell Tube Formation Protocol - http://www.bdbiosciences.com/pdfs/whitePapers/06-A790030-3A1.pdf

Category: Pharmacology and Toxicology Protocols: Drug Discovery

Protocol describes an assay that relies on the endothelial cells’ ability to form distinct blood-vessel-like tubules in an extracellular matrix where they can subsequently be visualized by fluorescence microscopy. Although quantification of the tubules can be performed by manual tracing, this method precludes the use of the assay in unbiased high-throughput applications. - [Read Image Based Assay of Endothelial Cell Tube Formation Protocol]

Imaging Actin in Tissue Slices from Transgenic Mouse Brain Protocol - http://www.cshprotocols.org/cgi/content/extract/2007/3/pdb.ip28

Category: Cytoskeleton Protocols: Actin Myosin Protocols

The same GFP-tagged actin construct used in cell transfection experiments has been used to produce transgenic mice. Transgenic animals allow the imaging of brain tissue in the intact animal, as acutely cut slices or as organotypic slice cultures. They also serve as a source of cells for imaging neurons at high resolution in dispersed low-density cell culture. In contrast to cells transfected in culture, where the level of actin-GFP expression in neurons varies considerably, transgenic mice... - [Read Imaging Actin in Tissue Slices from Transgenic Mouse Brain Protocol]

Immunohistochemistry with AP-Conjugated (NBT/BCIP) Protocol - http://icg.cpmc.columbia.edu/cattoretti/Protocol/immunohistochemistry/NBTBCIPstaining.html

Category: Immunohistochemistry Protocols

Protocol for immunohistochemistry with AP-Conjugated (NBT/BCIP). Protocol extensively blocks slides, further diluting the primary antibody, lengthening the incubation and washing time, using a simple AP-conjugated secondary at high dilution and use a slow long development with the most powerful IHC development, NBT/BCIP. Includes: Single AP stainiing and Double AP staining. - [Read Immunohistochemistry with AP-Conjugated (NBT/BCIP) Protocol]

Immunoprecipitation TroubleShooting Guide Sigma - http://www.sigmaaldrich.com/img/assets/8181/ImmunoprecipTroubleshoot.pdf

Category: Protein Protocols: Immunoprecipitation Protocols: Immunoprecipitation Troubleshooting

Immunoprecipitation TroubleShooting Guide. Sigma Aldrich. Problems: No binding, High background or unwanted proteins precipitate. With Solutions. - [Read Immunoprecipitation TroubleShooting Guide Sigma]

In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with DpnI - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3813

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for in vitro mutagenesis using double-stranded DNA templates. Two oligonucleotides are used to prime DNA synthesis catalyzed by a high-fidelity thermostable polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and occupy the same starting and ending positions on opposite strands of the plasmid DNA. - [Read In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with DpnI]

Induction of Conjugation in Tetrahymena Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4499

Category: Tetrahymena Protozoa Protocols

Mature Tetrahymena cells of opposite mating types are starved under appropriate salt conditions. The mating types are then combined to costimulate through cell-cell interaction. Loose pairs and then firm, irreversible pairs of cells of opposite mating types form. This method consistently results in a high percentage of pairing (usually greater than 80%) and good synchrony. - [Read Induction of Conjugation in Tetrahymena Protocol]

Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E6634D5A5D8B91EC631DE5426302733&objectid=6674D9B6A53E5856FBF23C4E94630203

Category: Immunology: T Cell Protocols

Describes methods for labeling high or low numbers of lymphocytes with CSFE. Protocols are provided to use CSFE-labeled cells in cell transfer studies or as cells to be cultured in vitro. Detailed guidelines for positioning of CSFE-labeled lymphocytes in lymphoid organs or other tissues are included for those wishing to use this approach to study lymphocyte migration. - [Read Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation]

Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4009

Category: PCR Protocols: PCR Cloning Protocols

Protocol describes the use of vectorette PCR and single-site PCR to amplify the terminal sequences of genomic sequences cloned in high-capacity vectors such as PACs and YACs. - [Read Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors]

Isolation and Purification of Tetrahymena Nuclei Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4500

Category: Tetrahymena Protozoa Protocols

Protocol describes how to allow the isolation of nuclei from all stages of the Tetrahymena life cycle in high yield with a high degree of purity. This method gives highly purified populations of both micronuclei and macronuclei. - [Read Isolation and Purification of Tetrahymena Nuclei Protocol]

Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4036

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Method of choice when large amounts of mammalian DNA are required, for example, for Southern blotting (Rapid Isolation of Mammalian DNA, Rapid Isolation of Yeast DNA, Southern Blotting: Capillary Transfer of DNA to Membranes) or for construction of genomic libraries in bacteriophage {lambda} vectors. Approximately 200 µg of mammalian DNA, 100-150 kb in length, is obtained from 5 x 107 cultured aneuploid mammalian cells (e.g., HeLa cells). - [Read Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol Protocol]

Labeling Antibodies with Biotin Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4286

Category: Antibody Protocols: Antibody Labeling Protocols

This protocol describes the covalent coupling of antibodies to biotin. Biotin groups bind with extremely high affinity to streptavidin and avidin, both of which are available commercially coupled with enzymes, fluorescent dyes, or iodine. A biotinylated primary antibody, therefore, can be detected with any of a wide variety of different labels. The biotinylation reaction is simple and mild, and rarely inactivates the antibody. - [Read Labeling Antibodies with Biotin Protocol]

Articles (1-7 of 7)

Genomic DNA Labeling of Microarrays Protocol

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 Polymerization Protocol using GTP and GMPCPP

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 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.

Immobilized Antigen Phage Antibody Selection Protocol

A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.

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.

Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.

Vector Design Protocol for Transgenic Mice Generation

The protocol gives general considerations for the design of targeting vectors for transgenic mice. The protocol shares tips in the design of knock-out and knock-in vectors and some of their strategies for producing homologously recombined embryonic stem cells.

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