nature Protocols

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. - [Read Chromatin Immunoprecipitation and Microarray-Based Analysis of Protein Location Protocol]

Category: Protein Protocols: Protein Phosphorylation Protocols

Fluorescent indicators for imaging protein phosphorylation in single living cells. Using Phocuses, genetically encoded fluorescent indicators, one can visualize signal transduction based on protein phosphorylation in living cells. Moritoshi Sato1, Takeaki Ozawa1, Kouichi Inukai2, Tomoichiro Asano2 & Yoshio Umezawa1. Nature Biotechnology - [Read Fluorescent indicators for imaging protein phosphorylation in single living cells]

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]

Category: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation of mRNA-protein complexes. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. - [Read Immunoprecipitation of mRNA-Protein Complexes Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Live-cell imaging techniques provide critical insight into the fundamental nature of cellular & tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein & synthetic fluorophore technology. Because of these advances, live-cell imaging has become a requisite analytical tool in most cell biology labs. Includes: Maintaining Live Cells on the Microscope Stage; Live-Cell Imaging Culture Chambers; Optical System and Detector Requirements etc. - [Read Introduction to Live-Cell Imaging Techniques]

Category: Signalling Signal Transduction Protocols: MAPK Protocols

Nature protocol entailing a kinase assay. - [Read Kinase Assays]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, etc. Protocol provides a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1–1.5 h. - [Read Laser-capture Microdissection Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Live-cell imaging techniques provide a critical insight into the fundamental nature of cellular and tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein and synthetic fluorophore technology. Because of these advances, live-cell imaging has become a requisite analytical tool in most cell biology laboratories. - [Read Maintaining Live Cells on the Microscope Stage]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for the optimization of elution condition for dye-ligand affinity chromotography. Elution methods used in dye-ligand affinity chromatography may be either selective or nonselective in nature.  Usually, selective elution methods are applied in combination with group-specific adsorbents, such as dye-ligand adsorbents, and nonselective elution methods are used in combination with highly specific adsorbents. - [Read Optimization of Elution Conditions for Dye-Ligand Affinity Chromatography Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

In preparation for FLIM-FRET analysis, the appropriate donor and acceptor components must be introduced into live or fixed cells. The method of introduction depends on the nature of the components and the state of the cells. For example, plasmid DNAs encoding a protein of interest fused to a variant of GFP may be introduced into live cells by transfection or microinjection, whereas labeled antibodies are delivered by microinjection. - [Read Probing Protein Interactions Using GFP and FRET Protocol]

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 19:971–974, 2001). NIH Stem Cell Unit - [Read Subcloning Protocol ES Cells]

Category: Protein Protocols: Protein Extraction From Tissue

Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics. Although many methods exist for fractionating proteins, the method described here can capture the majority of subcellular fractions simultaneously at reasonable purity. The scalability of this method makes it amenable to small samples, such as embryonic tissues, in addition to larger tissues. The protocol described is for the general fractionation and extraction of proteins from organs / tissue - [Read Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomic]

Category: Protein Protocols: Protein Electrophoresis

Tricine–SDS-PAGE Protocol and background. Nature. PDF file. Tricine–SDS-PAGE is commonly used to separate proteins in the mass range 1–100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. –SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification. - [Read Tricine–SDS-PAGE Protocol PDF]