Eccles:ProteinLysatesfromCellsinCulture
Cell Lysis Buffer5mL 0.1M Tris HCl pH 8 (10mM)0.44g NaCl (150mM)0.02g EDTA (1mM)0.5mL nonidet P40 (1% w/v)0.05g SDS (0.1% w/v)Make up to 50mL with MQH2O and filter sterilise.Store at 4 degrees C.This cell lysis buffer has a limited shelf life (at 4 degrees C). I recommend making fresh buffer up annually. My own personal experience has been that after ~18months my protein lysates began appearing very distorted followi......阅读全文
Eccles:Protein－Lysates－from－Cells－in－Culture
Cell Lysis Buffer5mL 0.1M Tris HCl pH 8 (10mM)0.44g NaCl (150mM)0.02g EDTA (1mM)0.5mL nonidet P40 (1% w/v)0.05g SDS (0.1% w/v)Make up to 50mL with MQH
Eccles:Protein－Lysates－from－Tissue
Cell Lysis Buffer5mL 0.1M Tris HCl pH 8 (10mM)0.44g NaCl (150mM)0.02g EDTA (1mM)0.5mL nonidet P40 (1% w/v)0.05g SDS (0.1% w/v)Make up to 50mL with MQH
Isolation－and－Culture－of－Multipotent－Stem－Cells－from－Human－Bone－Morrow
Bone marrow contains three types of stem cells:1. Hematopoietic stem cells give rise to the three classes of blood cells that are found in the circu
CO2恒温摇床解决人胚肾－293－(HEK293)－细胞结团问题（二）
Lysate preparation and western blottingProtein lysates were created by harvesting the cells from confluent T-flasks or from suspension cultures at h
使用CO2恒温摇床解决人胚肾－293－(HEK293)－细胞结团问题
人胚肾 293 (HEK293) 细胞在重组蛋白表达中是最常见的宿主细胞。 这类细胞能够表达大量的膜蛋白，如 G 蛋白偶联受体 (GPCR) ，是无法在最常见的生物制药生产宿主，如：中国仓鼠卵巢 (CHO) 细胞中作表达。 HEK293 虽然是蛋白表达的极好宿主，然而 HEK293 细胞
Culture－of－BEND－Cells－(Bovine－Endometrial－Cells)
Culture of BEND Cells (Bovine Endometrial Cells)Charles E. Krininger, III and Peter J. Hansen Dept. of Animal Sciences, University of FloridaThis prot
Cell－Extraction－Protocol
实验概要Primary tissues are valuable tools for the study of intracellular and extracellular markers which characterize disease states. We have developed
Decontamination－of－cells－from－the－yeast
I Destroy yeast1. Aspirate medium and wash cell in PBS.2. Incubate cells at 37oC for 5 min in non-diluted antibiotic-antimycotic.3. In
Chemical－Induction－of－Apoptosis
Chemical Induction of Apoptosis - 1 May 2001p53, p21WAF1, Myc, Bcl-2, Bax, Bcl-x and bak are among the proteins involved in the regulation of apoptosi
Isolation,－Culture,－and－Differentiation－of－Progenitor－Cells
Isolation, Culture, and Differentiation of Progenitor Cells from the Central Nervous SystemScott R. Hutton and Larysa H. Pevny1UNC Neuroscience Center
Isolation－and－culture－of－porcine－muller－cells
1. Porcine eyes were harvested from pentobarbital-anesthetized animals, each weighing 30 to 40 pounds, and transported to the laboratory in ice-co
Immunofluorescence－Microscopy－of－tissue－culture－cells
Immunofluorescence Microscopy of tissue culture cellsThese methods are written for direct staining of filamentous actin with bodipy FL-phallicidin and
Isolation－and－culture－of－pancreatic－stellate－cells
Pancreatic stellate cells (PaSCs or PSCs) are myofibroblast-like cells found in the areas of the pancreas that have exocrine function. PaSCs are
Culture－of－retinal－endothelial－cells－and－pericytes
Isolation of retinal microvessels1. Eyes were obtained and transported on ice.2. Eyes were cut circumferentially 3 mm posterior to the limbus, the v
Removing－cells－from－liquid－nitrogen
Put cryovial straight from storage and float in the 37篊 water bath- caution should be taken as on rare occasions vials can explode when heated up due
Dissociation－of－Cells－from－Primary－Tissue
实验概要A common method to obtain single cell suspensions from primary tissue is enzymatic disaggregation. Expose the cells to enzymes for a minimal am
Harvesting－Hematopoietic－Cells－from－Mice
Materials4 mice from each genotype4 Ly5 miceBuckets with wet ice 3xBucket with dry ice 1xDewar flask with liquid nitrogen100 mL beakers with 95% ethan
Fusion－Protein－Isolation(融合蛋白分离纯化)
Peter Novick Lab,Department of Cell Biology Yale University School of Medicinehttp://info.med.yale.edu/cellbio/Novick/Second/Protocols/Fusion.pdf1.Sta
Immunoprecipitation...－（一）
实验概要We provide a general IP procedure including a list of reagents and a table to help you choose the correct protein beads.Immunoprecipitation is a
同位素法测定底物磷酸化活性方法－Phosphorylation－of－Substrates
Phosphorylation of SubstratesScott T. Eblen, N. Vinay Kumar, and Michael J. WeberDepartment of Microbiology and Cancer Center, University of Virginia
Protein－extraction－from－whole－tissues－for－IEF
Modified from that of Jay Thelen - University of Missouri-ColumbiaPhenol extraction followed by methanolic ammonium acetate precipitation - an effecti
Isolation－and－Culture－of－Human－Liver－Stem－Cells
Materials and Methods1. Human hepatocytes were isolated from fresh surgical specimens of patients undergoing hepatectomies. Healthy liver tissue (
Culture－of－human－venous－smooth－muscle－cells
Culture of human venous smooth muscle cells 1. Explants of the 2 groups of skins (control subjects and patients with varicose veins) were prepared
Culture－of－Human－Embryonic－Stem－Cells－(hESC)
All cell lines are initially grown according to the supplier's protocols but we are adapting them to one simple protocol outlined below:6-well pla
Tissue－Culture－of－PtK1－cells
Please note: lately (2005 - present), we normally culture PtK1 cells in F-12 media, but they also grow in other types of media as described below.Mixi
Derivation－and－Culture－of－Dopaminergic－Neurons－(from－Midbrains－of－Rodents)
实验概要Dopaminergic (DA) neurons are located in the ventral midbrain (VM). The ability to isolate precursor cells and neurons from the VM provides a po
RNA－Isolation－From－Animal－tissue－or－cell－culture
实验概要This method is designed for most animal tissues and culture cells. For RNA isolation from fibrous tissue, follow the specialized protocol on pag
同位素法测定底物磷酸化活性方法
实验概要Ideally, one would like to be able to directly phosphorylate substrates in an intact cell. This could potentially be performed by introducing AT
Signaling－Pathway－from－GProtein－Families
G-aS-coupled receptors stimulate adenylyl cyclase (AC), which synthesizes cAMP from ATP. In contrast Gai-coupled receptors inhibit AC and so reduce cA
Isolation－and－Culture－of－human－vascular－smooth－muscle－cells
1. Human vascular smooth muscle cells (SMCs) were isolated from human saphenous vein.2. Small moistened pieces of tissue (6 mm2) were placed intima