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Media for Culture of Mammalian Cells Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E662B6F09C1BB8FBC47FC2A7E61A8A3&objectid=66739B890A55AC13354418E67F28E693

Category: Cell Biology and Cell Culture: Cell Culture Growth Media: Mammalian Cell Culture Growth Media Protocols

The culture medium is an essential component of the in vitro environment and must be selected or designed with care. This protocol provides guidelines for design of serum-containing and serum-free media, selective and specialty media, and media for growth under special conditions such as soft-agar growth. - [Read Media for Culture of Mammalian Cells Protocol]

Metabolic Labeling & Immunoprecipitation Protocols - http://www.cbrinstitute.org/labs/springer/protocols/jun_35Slabeling_IP.html

Category: Immunology: Immunoprecipitation Protocols: Immunoprecipitation of Radiolabeled Cells Protocols

Cells incorporate 35S-methionine or cysteine during the protein synthesis. Thus it is essential to use Met,Cys-free medium and dialyzed FCS during the labeling. Short period of incubation with 35S-methionine or cysteine will result in radiolabeling (pulse), and additional incubation with excess concentration of unlabeled Met+Cys (chase) is needed for complex glycoproteins like integrins to get expressed as a maturated form. - [Read Metabolic Labeling & Immunoprecipitation Protocols]

Nucleic Acid Hybridization General Aspects - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_33-37.pdf

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Discusses the effects of various components of the hybridization solution on the rate of renaturation and thermal stability of DNA hybrids free in solution. Includes: The main parameters that influence hybridization; Additional hybridization variables; Competition in situ hybridization; Oligonucleotide hybridization; Standard in situ hybridization conditions. - [Read Nucleic Acid Hybridization General Aspects]

Order Fluorophore Wall Chart - http://microscopy.bio-rad.com/fluorescence/fluorochrome.htm

Category: Molecular Imaging

Order Fluorophore Wall Chart. Free. CellScience - [Read Order Fluorophore Wall Chart]

Placenta Specific Gene Manipulation by Transducing Zona-Free Blastocyst using Lentiviral Vector - http://www.natureprotocols.com/2007/01/24/placenta_specific_gene_manipul.php

Category: Cytogenetics Protocols

Details a placenta specific gene manipulation by transducing blastocysts with lentiviral vectors1. After a removal of zona pellucida which functions as a physical barrier, trophoblast cells lying outermost layer of blastocyst were transduced from outside with high-titer lentiviral vectors. As most placental cells descend from trophoblast cells while fetus originated from inner cell mass, transgene expression can be observed in trophoblast cells from preimplantation stages and in placenta... - [Read Placenta Specific Gene Manipulation by Transducing Zona-Free Blastocyst using Lentiviral Vector]

Preparation of a Highly Efficient Transcription Extract from Drosophila Embryos Protocol - http://www.bio.com/protocolstools/protocol.jhtml%3Bjsessionid%3D3APITJJIWFIM1R3FQLMCFEWHUWBNSIV0?id=p9052

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol describes how to produce a soluble nuclear extract rich in basal pol II transcription factors from Drosophila embryos. This is a cell-free extract that contains all the necessary transcription factors and is capable of accurate initiation of transcription by RNA polymerase II but is deficient in core histones and histone H1. - [Read Preparation of a Highly Efficient Transcription Extract from Drosophila Embryos Protocol]

Preparation of Rabbit Reticulocyte Lysate Cell-Free System Protocol - http://www.bio.com/protocolstools/protocol.jhtml?id=p308

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for the preparation of rabbit reticulocyte lysate cell-free system. Protocol includes information on: Solutions; BioReagents and Chemicals and Protcol Hints. - [Read Preparation of Rabbit Reticulocyte Lysate Cell-Free System Protocol]

Protein Ligand Affinity Chromatography Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4203

Category: Chromatography Protocols: Affinity Chromatography Protocols

Protocol describes the coupling of peptides or proteins to Affi-Gel 10 (Bio-Rad). Affi-Gel 10 is a cross-linked agarose derivatized with N-hydroxysuccinimide (NHS) ester on a 10-atom-long spacer arm. The NHS ester allows spontaneous coupling of proteins via free amino groups. - [Read Protein Ligand Affinity Chromatography Protocol]

Protocol for Preparation of DNA-free RNA Prior to RT-PCR Fermentas - http://www.fermentas.com/techinfo/modifyingenzymes/protocols/p_prepdnafreerna.htm

Category: RNA Protocols: RT-PCR and cDNA synthesis

Protocol for Preparation of DNA-free RNA Prior to RT-PCR (with Deoxyribonuclease I) Fermentas - [Read Protocol for Preparation of DNA-free RNA Prior to RT-PCR Fermentas]

Protocols for the Maintenance of Human Embryonic Stem Cells in Feeder Free Conditions - http://www.geron.com/PDF/scprotocols.pdf

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

Includes protocols: Mouse Embryonic Fibroblasts (MEF) Preparation; Harvesting MEFs; Cryopreservation of MEFs; Thawing and maintaining MEFs; Irradiating & Plating MEFs; Culture of Human ES cells with Matrigel® and Conditioned Medium; Preparation of Conditioned Medium (CM); Preparation of Matrigel® -coated plates; Passage of human ES cells on Matrigel®; Daily maintenance of feeder-free culture; Freezing Human ES Cells; Thawing Human ES cells; Formation of Embryoid Bodies; - [Read Protocols for the Maintenance of Human Embryonic Stem Cells in Feeder Free Conditions]

Purification of Bacteriophage lambda Particles by Isopycnic Centrifugation through CsCl Gradients - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3968

Category: Viral Protocols

Isopycnic centrifugation through CsCl gradients is used to prepare infectious bacteriophage {lambda} particles of the highest purity that are essentially free of contaminating bacterial nucleic acids. - [Read Purification of Bacteriophage lambda Particles by Isopycnic Centrifugation through CsCl Gradients]

Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3655

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant proteins, constructed in pGEX vectors, are fused to glutathione S-transferase (GST) and can be purified to near homogeneity by affinity chromatography on glutathione-agarose. Bound GST-fusion proteins are readily displaced from the column by elution with buffers containing free glutathione. - [Read Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose Protocol]

Recycling Tubulin Protocol - http://mitchison.med.harvard.edu/protocols/recycle.html

Category: Cytoskeleton Protocols: Microtubules Protocols

Recycle tubulin fractions stored at -80¡C after the PC column and store the recycled tubulin in small aliquots for day-to-day use. Generally store recycled tubulin in Injection Buffer (IB) without free GTP. This is done because depolymerization appears to be much better in IB, IB is ideal for microinjections/adding tubulin to extracts, and the absence of free GTP makes polymerization with GMPCPP, a very useful GTP analog that has ~5-10X lower affinity than GTP for tubulin. - [Read Recycling Tubulin Protocol]

Reverse Transcriptase In Situ PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4119

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

This protocol describes a method for reverse transcriptase (RT) in situ PCR. In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step. The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in RNase-free DNase that is performed after protease digestion, and an RT step, prior to in situ PCR. - [Read Reverse Transcriptase In Situ PCR Protocol]

Reverse Transcriptase In Situ PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4119

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol describes a method for reverse transcriptase (RT) in situ PCR. In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step. The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in Rnase-free Dnase that is performed after protease digestion, and an RT step, prior to in situ PCR. - [Read Reverse Transcriptase In Situ PCR Protocol]

RNA Isolation and RT-PCR from dsRNA-Treated Mouse Oocytes and Early Embryos Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4513

Category: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes the use of RT-PCR to confirm knockdown of a gene of interest in RNAi-treated mouse oocytes and early embryos. All of the solutions used during RNA isolation and reverse transcription must be Rnase-free - [Read RNA Isolation and RT-PCR from dsRNA-Treated Mouse Oocytes and Early Embryos Protocol]

Scion Image for Mac and PC - http://www.scioncorp.com/frames/fr_download_now.htm

Category: Molecular Imaging

A free PC version of Image, Scion Image for Windows, is - Scion Corporation. - [Read Scion Image for Mac and PC]

Staining Resin Sections of Bone Protocol - http://www.nottingham.ac.uk/pathology/protocols/resbonestain.html

Category: Immunohistochemistry Protocols: Resin Staining Protocols

Protocol for staining resin sections of bone. Includes: VON KOSSA/VAN GIESON; GOLDNER TRICHROME (MODIFICATION); TOLUIDINE BLUE; PERLS PRUSSIAN BLUE; SECTIONS LABELLED WITH TETRACYCLINE; FLATTENING FREE-FLOATING SECTIONS; MOUNTING FREE-FLOATING SECTIONS; CLEANING SLIDES. - [Read Staining Resin Sections of Bone Protocol]

Steroid Radioimmunoassay Protocol - http://faculty.washington.edu/jwingfie/protocols.htm

Category: Immunology: Immunoassays

Protocol for steroid radioimmunoassay. Includes: SOLVENT DISTILLATION; PREPARATION OF PLASMA SAMPLES; EXTRACTION OF STEROIDS AND COLUMN PACKING; COLUMN CHROMATOGRAPHY; RADIOIMMUNOASSAY; SEPARATION OF BOUND AND FREE COUNTS; DIRECT ASSAYS; SHORT COLUMN CHROMATOGRAPHY. - [Read Steroid Radioimmunoassay Protocol]

Subcloning Protocol ES Cells - http://stemcells.nih.gov/research/NIHresearch/scunit/subcloning.asp

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture

Subcloning Protocol ES Cells. Used an adapted form of the feeder-free protocols detailed in Xu et al. (Nature Biotechnology 19:971â€“974, 2001). NIH Stem Cell Unit - [Read Subcloning Protocol ES Cells]

Subfractionation of Low-Density Lipoprotein (LDL) - http://www.axis-shield.com/densityhome/optiprep/M11.pdf

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

In the routine method described in this protocol, chylomicron-free plasma is adjusted to 12% (w/v) iodixanol and the sample, essentially fills an approx 3 ml tube for a near-vertical rotor. During the centrifugation VLDL, LDL and HDL particles and also plasma proteins migrate from all parts of the sample to their final buoyant density banding position in the self-forming density gradient. - [Read Subfractionation of Low-Density Lipoprotein (LDL)]

Articles (1-7 of 7)

Drosophila Cell Transfection Protocol

A simple Drosophila tissue culture cell transfection method.

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.

DNA Fragment Purification from Polyacrylamide Gels Protocol

A simple protocol for the purification of DNA fragments from Polyacrylamide Gels.

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.

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.