细胞功能测定

Immunofluorescence Staining of Human Cells by Lysed Whole Blood MethodAdd 100 m l of well-mixed anticoagulated whole blood to the bottom of a labeled tube. Add the appropriate primary antibody to each ...

General Annexin V Staining ProcedureSolutions 1. 10X Binding Buffer (Cat. No. 66121A) : 0.1 M HEPES pH 7.4; 1.4 M NaCl; 25 mM CaCl 2 . Dilute to 1X prior to use. 2. Propidium Iodide (PI). Prepare a ...

Fluorescence Procedures for the Actin and Tubulin Cytoskeleton in Fixed CellsActin: Louise CramerTubulin: Arshad DesaiGeneral StrategyWe typically work with tissue culture primary mammalian cells and ...

Flow Cell Assays with Microtubules: Motility/Dynamics in Fluorescence and VE-DIC Flow cell assays are very useful for studying microtubule motility microtubule dynamics kinetochore-microtubule interac ...

Labeling Tubulin and Quantifying Labeling StoichiometryThis is a general procedure for coupling moieties with reactive succinimidyl esters to tubulin. We have used it successfully to derivatize tubuli ...

Negative Stain Electron Microscopy of Microtubules Negative staining is a rapid qualitative method for analyzing microtubule structure at the EM level. Because negative staining involves deposition of ...

SDS Gel Electrophoresis of Tubulin\MAPsLEVEL II Figure 9.3 SDS-PAGE of tubulin and associated proteins Materials Stock Acrylamide: (30%T:0.8%C)30% by weight of acrylamide0.8% by weight of NN'-bis-meth ...

TEM Visualization of MicrotubulesLEVEL II Materials Coated grid for TEM0.1 M ammonium acetate5% ethanol saturated uranyl acetateTransmission electron microscope Procedure At the conclusion of a 37° C ...

Preparation of Segmented and Polarity Marked Microtubules Segmented and polarity-marked microtubules are very useful for many different types of in vitro assays. Segmented microtubules are microtubule ...

Subculturing CellsMaterials1. Medium with supplements (serum glutamine antibiotics). 2. STE 37oC. Stored at 4oC. 3. Trypsin 0.25% or 0.05% 37oC. Stored at -20 oC. Do not leave trypsin in waterbath for ...

Cell Disruption and Subcellular FractionationContributor: Suprya JayadevDate: July 31 1991Nitrogen Bomb1) Harvest cells and wash 2 times with PBS.2) Resuspend final pellet in relaxation buffer. (20 ml ...

CHO Centrosome Prep:Arshad Desai4/94Cells:We grow our CHOs with MEM (without nucleosides) + 10% Bovine Calf Serum and penn/strep/glutamine. For a prep it is best to grow twenty large plates (150 mM) a ...

CSF Extract Prep for Spindle AssemblyClaire Walczak11/95This protocol is essentially as described by Murray (1991) Cell Cycle Extracts. In Methods in Cell Biology B.K. Kay and B. Peng eds. (San Diego: ...

Large Scale Tubulin Preparation Tubulin is purified from bovine/porcine brain by two cycles of polymerization/depolymerization followed by removal of copurifying proteins on a phosphocellulose (PC) co ...

Lysosome Isolation in Isotonic SucroseLEVEL I MATERIALS Rat liverPhysiological saline (0.85% w/v NaCl)0.25 M sucrose in 10 mM Tris-HCL pH 7.4Brendler teflon homogenizerRefrigerated preparative centrif ...

These protocols should yield enough cytosol and organelles for 1-200 MT/Organelle motility assays.Solutions and Reagents Freshly removed or flash frozen rat liver PBS Homogenization Buffer Homogeniz ...

Organelle DNA Library Construction(version MAY-1998)I. NEBULIZATION of DNA 1. 0.5 - 5 ug DNA in TE (10mM/1mM) 25% glycerol final volume 500 ul 2. nebulize for 90-100 sec at 5-10 psi (for a med ...

Preparation of Mitochondria from Rat Liver Rat liver is an ideal source for functional intact mitochondria for a number of reasons. We use Sprague-Dawley albino male rats for our studies. Female rats ...

Recommended Experiments with Isolated Mitochondria Each oxygraph setup will consist of two stations each with dissolved oxygen chamber and chart recorder connected to a two-channel oxygen monitor. Ple ...

Mitochondria Structure and Function - Theoretical DetailsIn an attempt to be concise and understandable introductory level courses and textbooks frequently present concepts that are technically correc ...