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Adaptation of High Five™ Cells to HyQ© SFX Insect Medium - http://www.hyclone.com/pdf/procedure_insect_highfive.pdf

Category: Cell Culture Protocols: Insect Cell Culture Protocols

This protocol is designed to adapt High Five™ (BTI-Tn-4) cells to serum-free suspension culture in HyQ SFX-Insect in 5 passages. The entire adaptation process takes approximately two weeks and the resulting adapted cells have doubling times of 16 and 20 hours and minimal clumping in suspension culture. - [Read Adaptation of High Five™ Cells to HyQ© SFX Insect Medium]

Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000230.pdf?pid=PP00000230

Category: RNAi Technology Protocols

Protocol describes the preparation of an shRNA-expressing Gateway entry vector. This process is preceded by the design and synthesis of target gene-specific sense and antisense oligonucleotides which, when annealed, will constitute an shRNA cassette. - [Read Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol]

Applications of SKY in Cancer Cytogenetics - http://www.utoronto.ca/cancyto/protocols_software/cytogenetics/SKY.html

Category: Cytogenetics Protocols: Spectral Karyotyping SKY Protocols

SKY has been applied to various tumor groups including hematological malignancies, sarcomas, carcinomas and brain tumors, with the intent of identifying specific chromosomal abnormalities that may provide insight to the genes involved in the disease process as well as identifying recurrent cytogenetic markers for clinical diagnosis and prognostic assessment. - [Read Applications of SKY in Cancer Cytogenetics]

Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol - http://www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4450

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo. - [Read Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol]

C. elegans Gene Knockout Protocol - http://www.zoology.ubc.ca/~alorch/koprotocol.htm

Category: C. elegans Protocols: C. elegans Genetics Protocols

The procedure is to mutagenize a large population of worms with trimethylpsoralen and UV irradiation, set up 1152 subpopulations, screen DNA made from this library for deletions in specific genes by nested PCR, and then to recover single worms carrying the deletions through a sib-selection process. - [Read C. elegans Gene Knockout Protocol]

Construction of Micro-Tip for Use in IMAC Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000163.pdf?pid=PP00000163

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the second step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LCMS/ MS. This procedure describes the construction of microchromatographic columns, or micro-tips. - [Read Construction of Micro-Tip for Use in IMAC Protocol]

Cosuppression in C. elegans Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4318

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Cosuppression is a process in Caenorhabditis elegans that closely resembles RNAi. In contrast to RNAi, however, the cosuppression effect in C. elegans does not spread throughout the animal. Cosuppression in C. elegans can be triggered by highly repetitive transgenes that contain gene constructs. - [Read Cosuppression in C. elegans Protocol]

Cryogenic Preservation and Storage of Animal Cells Protocol - http://www.corning.com/Lifesciences/technical_information/techDocs/Cell_freezing_protocol.pdf

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

Cryogenic preservation (storage below -100°C) of cell cultures is widely used to maintain backups or reserves of cells without the associated effort and expense of feeding and caring for them. The success of the freezing process depends on four critical areas: Proper handling and gentle harvesting of the cultures; Correct use of the cryoprotective agent; A controlled rate of freezing; Storage under proper cryogenic conditions. - [Read Cryogenic Preservation and Storage of Animal Cells Protocol]

Cryosectioning Protocol - http://genetics.med.harvard.edu/~cepko/protocol/mike/C7.html

Category: Immunohistochemistry Protocols: Cryosectioning Protocols

Protocol for cryosectioning. While the timing of the various steps in this protocol are probably not critical, process the tissue all at once to ensure that RNA and/or proteins do not get degraded. Includes: 20% Paraformaldehyde/4% Paraformaldehyde-PBS; Sucrose/PBS. - [Read Cryosectioning Protocol]

Detection of apoptotic process in situ using immunocytochemical - http://www.cyto.purdue.edu/flowcyt/research/cytotech/apopto/data/barbie.htm

Category: Cell Biology and Cell Culture: Apoptosis Protocols

Detection of apoptotic process in situ using immunocytochemical and TUNEL assays. Barbieri et al. Purdue Cytometry CD-ROM Series - [Read Detection of apoptotic process in situ using immunocytochemical]

FAQs About Cell Sorting - http://cyto.mednet.ucla.edu/Protocols/question.htm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Sorting Protocols

Questions and answers about cell sorting. Includes: When should I use fluorescence activated cell sorting over bulk separation methods like panning or magnetic bead separations? Will my cells be harmed by the sorting process? How many cells do I need to prepare to recover 1 X 106 of a population that comprises 10% of the cells? Are there ways to improve sort recovery? etc... - [Read FAQs About Cell Sorting]

Fluorescent Reagents for Live Cell Imaging and Their Introduction into Cells - http://www.cshprotocols.org/cgi/content/abstract/2006/29/pdb.ip20

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Most biological specimens are relatively transparent, so details of internal and intracellular morphology are difficult to image in untreated living specimens using simple bright-field techniques. Fluorescence microscopy offers greater advantages and possibilities for increasing contrast and determining the specific localization of molecules in cells. Article outlines the three methods most commonly used to introduce an appropriate label into Drosophila tissue without perturbing the process. - [Read Fluorescent Reagents for Live Cell Imaging and Their Introduction into Cells]

Guide For Identifying And Correcting Common Cell Growth Problems - http://www.corning.com/Lifesciences/technical_information/techDocs/cc_guide_identifing_correcting_common_cell_growth_problem_5_2_03_cls_cc_013w.pdf

Category: Cell Biology and Cell Culture: Troubleshooting Cell Culture Problems

Guide For Identifying And Correcting Common Cell Growth Problems. Corning. Surface Treatment Process, Problems Related to Technique, Problems Related to Incubators, Problems Related to Culture Media, Problem Solving Suggestions. - [Read Guide For Identifying And Correcting Common Cell Growth Problems]

Isolation of Arabidopsis Nuclei and Measurement of Gene Transcription Rates Protocol - http://www.natureprotocols.com/2007/01/25/isolation_of_arabidopsis_nucle.php

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol for the isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Plant materials are ground in hexylene glycol-based buffers and highly enriched nuclear fractions are obtained using Percoll density gradients. Standard and small-scale protocols are presented, along with a tested method for nuclear run-on assays. The entire process may be completed within 3 days. - [Read Isolation of Arabidopsis Nuclei and Measurement of Gene Transcription Rates Protocol]

Lysis and Protein Extraction from WEHI-231 Cells with TriPure Isolation Reagent Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000162.pdf?pid=PP00000162

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the first step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LC-MS/MS. This procedure is used to prepare protein extracts from WEHI-231 cells. This preparation method provides total cellular protein samples that are free of contaminating nucleic acids. - [Read Lysis and Protein Extraction from WEHI-231 Cells with TriPure Isolation Reagent Protocol]

Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4449

Category: Transfection Protocols

Protocol describes transfection of plasmid DNA into mammalian cell lines using electroporation, a process whereby external application of electric pulses induce cell membrane permeability. Cells in suspension and small volume cells are difficult to transfect, whereas adherent cells and large volume cells are relatively easy. Regardless of cell size or phenotype, transfection efficiency increases with a high concentration of cells in a small volume. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4449

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol describes transfection of plasmid DNA into mammaliancell lines using electroporation, a process whereby externalapplication of electric pulses induce cell membrane permeability.A number of factors can affect electrotransfection efficiency.In general, cells in suspension and small volume cells are difficultto transfect, whereas adherent cells and large volume cellsare relatively easy. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Preparation and Enrichment of Phosphopeptides Using IMAC and LC-MS/MS - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000164.pdf?pid=PP00000164

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the third step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LCMS/ MS. - [Read Preparation and Enrichment of Phosphopeptides Using IMAC and LC-MS/MS]

Process Protocol for Hepatocyte Harvest - http://www.biolifesolutions.com/protocols/Hepatocyte_Protocol.pdf

Category: Cell Biology and Cell Culture: Hepatocyte Cell Culture

Process Protocol for Hepatocyte Harvest ... Re-suspend as necessary in culture media (for cell culture) or in CryoStor ... - [Read Process Protocol for Hepatocyte Harvest]

Protein Precipitation Microplate - http://www.htslabs.com/HTML/Wellplates/Applications/protein_precipitation_microplate.htm

Category: Protein Protocols: Protein Precipitation Procols

Laboratory sample cleanup is a necessary part for analytical preparation analysis. The removal of Contaminants such as proteins, cell debris and other materials is an important step. Typically this has been done by using Acetonitrile and then Centrifugation to pellet the debris leaving the clean supernant. After this process supernatant can be used for further analysis by HPLC, GC, MS and other analysis tandem methods. HTS Labs. - [Read Protein Precipitation Microplate]

The Liquid Scintillation Counting Process - http://laxmi.nuc.ucla.edu:8248/M248_99/autorad/Scint/scint1.html

Category: Radioactivity: Liquid Scintillation Counting

The Liquid Scintillation Counting Process. Great Review of the Process and detection procedure of the counter. Dr. Gambhir - [Read The Liquid Scintillation Counting Process]

Western Blot Analysis—Phosphoprotein-Specific Antibody Mixture Protocol - http://www.afcs.org/reports/v1/BC0001/Protocols/WBPhosphoSpecific.pdf

Category: Immunology: Immunoassays

3-step process of Western immunoblotting for detection of particular sites of phosphorylation on specific proteins recognized by modification-sensitive antibodies. - [Read Western Blot Analysis—Phosphoprotein-Specific Antibody Mixture Protocol]

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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.

Microinjection Pipette Pulling Protocol

A simple protocol for microinjection pipette pulling.

Paraffin Embedding Protocol

Paraffin Embedding Protocol for molecular profiling. This Paraffin Embedding Protocol describes the processing of the tissues into sections following ethanol fixation. Molecular profiling (MP) is a technique that is used to visualize the global patterns of RNA expression or protein expression in various cell types and disease processes.

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.

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.

Picking Transfected ES Cell Clones Protocol

This protocol a protocol on how to generate transfected embryonic stem (ES) cell clones. The previous protocol in this series is the Protocol for Electroporation of ES cells. The next protocol in the series is the Protocol on Disaggregation, Expansion, and Freezing of Transfected ES Clones.

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