Protocol describes methods to superinfect bacteria carrying a recombinant phagemid with a high-titer stock of an appropriate helper virus and to assay the yield of filamentous virus particles that carry single-stranded copies of the phagemid DNA. The key to success in using phagemids is to prepare a stock of helper virus whose titer is accurately known. - [Read Producing Single-stranded DNA with Phagemid Vectors Protocol]
Proliferative Assays for T Cell Function Protocols- https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E6634679E96E622003E15187F105306&objectid=6674B7F5E219FD4021F448C7D13AF27A
Protocols describe the conditions required to induce proliferation are described. Also describe the assay in which CD4·CD25·T cells are co-cultured with conventional T cells in order to assess their suppressive function. Will describe the culture conditions for the activation and expansion of CD4·CD25· cells. - [Read Proliferative Assays for T Cell Function Protocols]
Protein Kinase C Assay- http://cancer.ucsd.edu/newtonlab/Newton%20Web%20PKC%20ASSAY.html
Methods on protein trafficking. Information on N5 Secretion and protein trafficking. Methods include: Alcian Blue Test; Lucifer Yellow uptake assay; Preparation of total protein extracts for western immunoblots; Screen for Drug Sensitivity. - [Read Protein Trafficking Methods]
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 - [Read Protocol for Competitive Ligand Binding to Cortical Receptor using Crosslinking]
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 (STAT3N–/–, SOCS3N–/–). Procedure includes: Surgical procedures, Functional evaluation, Immunohistochemistry, In vitro migration assay. - [Read Protocol for Conditional Ablation of stat3/socs3 Discloses the Dual Role for Reactive Astrocytes]
Protocol describing a JNK kinase asssay. Protocol includes information on: Triton Lysis Buffer, Kinase Assay Buffer, Kinase Assay and Preparation of kinase - [Read Protocol for JNK Kinase Assay]
This protocol focuses on the interactions between L-selectin expressed on neutrophils and PNAd coated onto the plastic surface. The main purpose of the flow chamber assay is to visualize and measure interactions between flowing cells expressing a given adhesion molecule on their surface, and their receptor, either directly coated on the flow chamber lower wall or expressed on a cell monolayer. - [Read Protocol for L-selectin-PNAd Interactions under Flow Conditions.]
Protocol for luciferase assay for in vitro detection. Protocol includes: Before cell lysate preparation; Cell lysate preparation; Protocol for manual luminometers; Protocol for plate reading luminometer; Preparation of protein assay reagent; Protein standards. - [Read Protocol for Luciferase Assay for In Vitro Detection]
Protocol describes an RNA Polymerase III (Pol III) transcription assay using an extract or proteins of choice. Pol III is the polymerase responsible for transcribing 5S RNA, tRNAs, and other small RNAs. α-Amanitin inhibits Pol II transcription in the assay. The newly-transcribed, radiolabeled RNA is visualized by autoradiography following Urea Polyacrylamide gel electrophoresis. - [Read Protocol for Polymerase III In Vitro Transcription]
This protocol describes an in vitro reaction to assay mitotic spindle assembly. The assay uses Cytostatic Factor extract made from Xenopus eggs, fluorescently-labelled tubulin, and prepared sperm nuclei. Spindle assembly is monitored by immunofluorescence microscopy. - [Read Protocol Spindle Assembly In Vitro]
Serum concentrations of voriconazole should be measured in patients receiving this drug to ensure that therapeutic levels are being achieved. The assay will give an indication of whether suitable blood levels have been achieved. - [Read Protocol: Bioassay for Determining Voriconazole Levels in Blood]
The activity of ß-glucuronidase (GUS) can be accurately determined in intact plant tissue using 4-methylumbelliferyl ß-D-glucuronide (4-MUG) as a substrate. Upon hydrolysis by GUS, the fluorochrome 4-methyl umbelliferone (4-MU) is produced. This method is based on the permeability of both 4-MUG and 4-MU through plant tissue. It consists of incubation of the tissue with the reagent and quantification of the fluorescence emitted by 4-MU in the solution. GUS activity in each sample can be... - [Read Quantitative GUS Activity Assay in Intact Plant Tissue Protocol]
Using excitation at 365 nm and measuring emission at 455 nm, the amount of 4-MU produced can be quantified. Under these conditions, background fluorescence from the substrate is negligible, especially if the appropriate filter is selected. - [Read Quantitative GUS Activity Assay of Plant Extracts]
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.
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.
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.