Assay of Cyclic AMP in Lysates of Cells Protocol allows you to measure the content of cyclic adenosine 3',5'-monophosphate (cyclic AMP or cAMP) in splenic B lymphocytes (B cells) in an enzyme-linked immunoassay. This protocol utilizes acetylation of cAMP to improve sensitivity and reduce interference. Protocol includes information on: how to determine cAMP, calculations and reagents and materials.
Assay of Cytokines in Tissue Culture Supernatants Assay of cytokines in tissue culture supernatants describes a liquid suspension array for quantification of cytokines in tissue culture supernatants or serum. With this assay, it is possible to profile the level of multiple cytokines in a single well. The principle of this cytokine assay is similar to a capture sandwich immunoassay. Includes: Preparation for the Assay, Cytokine Assay, Reagents and Materials.
FAK Autophosphorylation Assay This kinase assay is meant to determine whether an agonist or event can influence the autophosphorylation of FAK. The addition of 1 Î¼l of polyGT to the kinase reaction mix will determine the activity of the enzyme against a substrate. Includes information on: Harvest, Immunoprecipitation, Kinase Reaction and Antibody Detection of FAK.
General Method for Ras, Rac, Cdc42, and Rho Activation Assay General protocol for Ras, Rac, Cdc42, and Rho activation assay. Includes: Affinity Precipitation/Immunoblot Protocol, Cell Culture and Extract Preparation (Adherent and Non Adherent cells), GTPÎ³S/GDP Loading for Positive and Negative Controls, Ras, Rac ,Cdc42, and Rho Pull-Down Assay and Western Blot and Detection.
Histone H1 Kinase Activity Assay 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. Includes information: H1 Kinase Assay on Individual Xenopus Oocytes; H1 Kinase Assay on Xenopus Egg Extract Samples; H1 Kinase Assay on Tissue Culture Cells; Helpful protocol hints.
In Vitro T Cell Activation Assess T cell activation protocol - measure T cell proliferation upon in vitro stimulation of T cells via antigen or agonistic antibodies to TCR. eBioscience
In-Gel Kinase Assay Protocol describes how to assay for kinase activity within a polyacrylamide gel, rather than in solution. The advantages to an in-gel assay are that an apparent molecular weight can be assigned to the kinase activity and that multiple kinase activities can be distinguished. Includes protocol hints.
Kinase Assay Using In Vitro Translated Xenopus Mos Kinase Protocol describes how tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay. Protocol includes: Translation of Xenopus Mos Kinase; Antibody to Antigen Binding; Protein A Sepharose to Antibody Binding; Kinase Reactions on Immunoprecipitated Material; Polyacrylamide Gel Analysis of Immunoprecipitates. Includes protocol hints.
Pheromone Halo Assay Protocol Pheromone Halo Assay. Dohlman Lab Protocol. An assay which measures the responsiveness and effects of cells to a factor pheromone.
Preparation of B-Lymphocyte Lysates for Cyclic AMP Determination This protocol provides a sufficient sample for several determinations of cAMP using the acetylation protocol. The method chosen for measuring the content of cyclic adenosine 3',5'-monophosphate (cyclic AMP or cAMP) in splenic B lymphocytes (B cells) is an enzyme linked immunoassay system. Protocol includes information on: Treatment of Cells and Preparation of Extracts; Reagents and Materials.
Preparation of Myocyte Lysates for Cyclic AMP Determination Protocol provides a method for acheiving a sufficient sample for several determinations of cAMP. The protocol described for measuring the content of cyclic adenosine 3',5'-
monophosphate (cyclic AMP or cAMP) in cardiac myocytes is an enzyme-linked immunoassay system. Protocol includes information on: Treatment of Cells and Preparation of Extracts; Use of Environmental Chamber; Reagents and Materials.
Preparation of RAW 264.7 Lysates for Cyclic AMP Determination The protocol provides a method to achieve a sample sufficient for one determination of cAMP using the acetylation protocol. The protocol describes the method used for measuring the content of cyclic adenosine 3',5'- monophosphate (cyclic AMP or cAMP) in RAW 264.7 cells using an enzyme-linked immunoassay system. Information included in the protocol: Treatment of Cells and Preparation of Extracts; Reagents and Materials.
Protocol for Antiphosphotyrosine Western Blot Analysis Detection of phosphorylated tyrosine residues can be performed using anti-P-TYR Ab and Western Analysis.Includes 2nd method,which uses phosphotyrosine in conjunction with anti-P-TYR Ab to "unlabel" potential proteins.By comparing Westerns developed with the 1st method(reveals phosphorylated protein) and the 2nd method(reveals non-specific labeling), a more accurate picture of those proteins phosphorylated on tyrosine can be seen. Includes: Protein Preparation, Electrophoresis and Transfer.
Protocol for Competitive Ligand Binding to Cortical Receptor using Crosslinking Protocol describes a competitive ligand binding assay for cortical neurotrophin receptors. Following binding in the presence of competitor, the bound radiolabeled ligand is cross-linked to the receptor. The cells are lysed and the ligand-receptor complexes are immunoprecipitated using a pan-trk (tyrosine kinase receptor) antibody. Protocol includes:Preparation of Cortical Tissue for Competitive Crosslinking, Competitive Binding, Crosslinking Ligand to Receptor, Lysis and Immunoprecipitation etc
Protocol for Conditional Ablation of stat3/socs3 Discloses the Dual Role for Reactive Astrocytes Protocol demonstrates that reactive astrocytes play a crucial role in wound healing and functional recovery by using mice with a selective deletion of the signal transducer and activator of transcription-3 (STAT3) or suppression of cytokine signaling-3 (SOCS3) under the control of Nestin gene promoter/enhancer (STAT3Nâ€“/â€“, SOCS3Nâ€“/â€“). Procedure includes: Surgical procedures, Functional evaluation, Immunohistochemistry, In vitro migration assay.
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.