assays Protocols

Category: Protein Protocols: Nuclear Extraction Protocols

Nuclear Extract protocol is optimized for about 10^7 cells, can use for gel shift assays and western blotting. Mirmira Lab, Univ. Virginia - [Read Preparation of Nuclear Extracts for Gel Shifts and Westerns Blots]

Category: Cytoskeleton Protocols: Microtubules Protocols

Segmented and polarity-marked microtubules are very useful for many different types of in vitro assays. Segmented microtubules are microtubules with a bright seed and dim elongated segments on both ends. Polarity marked microtubules are microtubules with a bright seed and a dim elongated segment only on one end -- the plus end. - [Read Preparation of Segmented and Polarity Marked Microtubules Protocol]

Category: Xenopus Protocols: Xenopus Egg Extracts Protocols

Protocol describes the preparation of nuclei from Xenopus sperm. These nuclei can be used for spindle assembly assays, microscopy, or DNA synthesis. - [Read Preparation of Xenopus Sperm Nuclei Protocol]

Category: Immunology: B Cell Protocols

Describes procedures for measuring the capacity of purified B cells to undergo proliferation. The method centers on the use of polyclonal stimulating agents (mitogens) because these agents stimulate the majority of B cells and because the alternative (measurement of antigen-induced proliferation) requires the laborious procedures of isolating antigen-specific B cells (which are otherwise present in too low a concentration in whole B cell populations). - [Read Proliferative Assays for B Cell Function Protocol]

Category: Immunology: T Cell Protocols

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]

Category: Protein Protocols: Protein Purification and Identification

Protein Purification: Assays, Specific Activity, Initial Fractionation. Blaber Lecture. - [Read Protein Purification: Assays, Specific Activity, Initial Fractionation]

Category: Cell Biology and Cell Culture

Microtubule binding assay protocol using the materials: Siliconized ultracentrifuge microfuge tubes, GTP-depleted microtubules, 6X SDS loading dye, 1X SDS loading dye, Coomassie Brilliant Blue R 250 (0.8% in 50% methanol + 10% acetic acid)and Destaining solution (13% methanol + 3% acetic acid). - [Read Protocol for Microtubule Binding Assays]

Category: Cell Biology and Cell Culture

Drosophila Larval Chemotaxis Assays. Information on: Larva harvesting; Olfactory tests on control larvae; Measuring response. - [Read Protocol for Drosophila Larval Chemotaxis Assays]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Cryopreserved PBMCs are a common specimen source for studies of immunological responses to vaccines, immunotherapies, etc. The health and viability of cells recovered post-cryopreservation are of course critical to the success and accuracy of immunological assays performed on them. This protocol standardizes PBMC isolation and cryopreservation techniques, specifically for the assessment of thawed cells by cytokine flow cytometry. - [Read Protocol for Isolation, Cryopreservation, and Thawing of PBMCs]

Category: Cell Biology and Cell Culture

Matirgel is considered as basement membrane and generated from EHS sarcoma. Matrigel contains not only basement membrane components (collagens, laminin, and proteoglycans)but also matrix degrading enzymes/their inhibitors and growth factors. Invasion of tumor cells into Matrigel has been used to characterize involvement of ECM receptors and matrix degrading enzymes which play roles in tumor progression. - [Read Protocol for Matrigel Invasion Assays]

Category: Cell Biology and Cell Culture

Purified Tubulin is polymerized and then stabilized with Taxol. The resulting Microtubules are then ready for use in motility assays. Protocol use solutions such as 50 mM Mg-GTP, 50 μl 100 mM GTP, 40 μM Taxol, Taxol Stock,and PM Buffer. - [Read Protocol for Microtubule Assembly]

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

Protocol includes procedure for Transwell in vitro migration assays. - [Read Protocol for Trans-Well Migration Assay]

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

In this protocol nuclei isolated from cells expressing the gene of interest are incubated with radiolabeled UTP which is incorporated into nascent RNA transcripts by RNA polymerase molecules that were actively transcribing at the time the cells were harvested. Because very little denovo initiation of RNA synthesis occurs in isolated nuclei, transcription of the target gene can be measured by hybridizing the radiolabeled RNA to an excess of the target gene immobilized on a nitrocellulose or nylon - [Read Protocol for Transcriptional Run- On Assays]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Protocol for purification of Lin-, c-kit+, Sca-1+ bone marrow cells for Culture, Flow Cytometry, or Transplantaion. Includes: Antibodies for Lineage Depletion; Harvest Marrow; Lineage Depletion; Cell Sorting; Viral Transduction; Transplant; CFU assays; GFP and cell surface marker immunostaining. - [Read Purification of Lin-, c-kit+, Sca-1+ Bone Marrow Cells for Culture, Flow Cytometry, or Transplantaio]

Category: Immunology

Protocol for purification of Lin-, c-kit+, Sca-1+ bone marrow cells for Culture, Flow Cytometry, or Transplantaion. Includes: Harvest Marrow; Lineage Depletion; Cell Sorting; Viral Transduction; Transplant; CFU assays; GFP and cell surface marker immunostaining. - [Read Purification of Lin-, c-kit+, Sca-1+ Bone Marrow Cells Protocol]

Category: DNA Protein Interactions Protocols: Electrophoretic Mobility Shift Assays EMSA Protocols

Protocol for radiolabeling of probes for electrophoretic mobility shift assays. - [Read Radiolabeling of Probes for Electrophoretic Mobility Shift Assays Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol for rat Chromaffin cells primary cultures: Standardization and quality assessment for single-cell assays. Includes: Preliminars; Isolation of the rat adrenal medulla; Enzymatic digestion of medullary tissue; Collecting and culturing the cells. - [Read Rat Chromaffin Cells Primary Cultures Protocol]

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols: Rat Neuronal Cell Culture Protocols

Protocol for standardization of both the isolation procedure and the quality of the cultures. - [Read Rat Chromaffin Cells Primary Cultures Standardization and Quality Assessment for Single Cell Assays]

Category: RNA Protocols: RNA-binding Protein EMSA Interactions

SULLY et al., 2004 Biochem. J. 377 (629–639). - [Read RNA EMSA (electrophoretic mobility shift assays) and UV cross-linking]

Category: ELISA Protocols: Sandwich ELISA Assay Protocols

Sandwich ELISA Protocol. Chemicon. General Protocol for the Sandwich ELISA method, Troubleshooting Sandiwich ELISA Assays. - [Read Sandwich ELISA Protocol]

Category: Signalling Signal Transduction Protocols

SAPK/Jun kinase assays. Preparation of cell lysate, Substrate/Antibody, Lysate Interaction and buffer recipes. Woodgett Lab PMH. - [Read SAPK / Jun kinase assays]

Category: Signalling Signal Transduction Protocols: MAPK Protocols

Protocol for SAPK/Jun kinase assays. Protocol includes information on: preparation of cell lysate, substrate/antibody, lysate interaction. - [Read SAPK/Jun Kinase Assays]

Category: Cell Biology and Cell Culture

This protocol describes how to isolate human platelets from whole blood. Isolated platelets are used for static adhesion assays, for flow chamber assays, flow cytometry measurements, etc. - [Read Separation of Platelets from Whole Blood]

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

Information for protocol using single-tube, coupled transcription/translation reactions for eukaryotic in vitro translation. Includes information on: Translation Procedure; Positive Control Translation Reactions Using Luciferase; Cotranslational Processing Using Canine Pancreatic Microsomal Membranes; Post-Translational Analysis; Positive Control Luciferase Assays; Composition of Buffers and Solutions; Luciferase SP6/T7 Control DNAs - [Read Single-tube Coupled Transcription/Translation Reactions for Eukaryotic In Vitro]

Category: Pharmacology and Toxicology Protocols: Drug Discovery

In Vitro ABC Transporter Assays for Hepato-Biliary DMPK; ABC transporter assays; Uptake transporter assays; Transporter assays for enteral absorption PK; Transporter Assays for Blood-Brain-Barrier PK; In Vitro Transporter Assays for renal excretion. - [Read SOLVO In Vitro Transporter Assay Technology]