This protocol assays inhibition of in vivo binding of [3H]-cAMP to cAR1 by GTPγS. Dictyostelium discoideum respond to extracellular cAMP through the cAMP chemoattractant receptor (cAR1). Binding of cAMP to the G protein-coupled cAR1 is inhibited by the GTP analog GTPγS. Protocol includes information on: Solutions used, BioReagents and Chemicals and Protocol Hints. - [Read GTPγS-Induced Inhibition of cAMP Binding to the cAMP Receptor (cAR1) in Dictyostelium Discoideum]
Protocol for in vitro transcription and translation using the coupled reticulocyte lysate system. This protocol is designed to test random samples on a protein gel. Scale up the reactions accordingly. Protocol includes: Procedure, Solutions, BioReagents and Chemicals and protocol hints. - [Read In Vitro Transcription and Translation Using the Coupled Reticulocyte Lysate System]
Protocol for preparation of KC nuclear extract for in vitro splicing. Protocol makes 3.4 ml of extract for every 4 liter of cells (depending on initial cell concentration). Protocol includes: Procedure, Solutions, BioReagents and Chemicals and protocol hints. - [Read Preparation of KC Nuclear Extract for In Vitro Splicing]
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.
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.
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.