LabelingTubulinandQuantifyingLabelingStoichiometry
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 tubulin with succinimidyl esters of biotin, digoxigenin, and a wide range of fluorochromes such as tetramethylrhodamine, X-rhodamine, fluorescein, Oregon Green, Cy3, Cy5 and C2CF (bis-caged carboxyfluorescein). The procedure i......閱讀全文
Labeling－Tubulin－and－Quantifying－Labeling－Stoichiometry
Labeling Tubulin and Quantifying Labeling StoichiometryThis is a general procedure for coupling moieties with reactive succinimidyl esters to tubulin.
Labeling－Tubulin－and－Quantifying－Labeling－Stoichiometry2
II. Labeling ProtocolThe procedure described below can be scaled down if desired. It is essential to perform all steps involving caged dyes under a sa
細胞組分和細胞器——細胞骨架
Fixation and Immunofluorescence of the Cytoskeleton?(Mitchison Lab)??Recycling Tubulin?(Mitchison Lab)??Labeling Tubulin and Quantifying Labeling Stoi
Tubulin－Preparat
Materials3 - 5 Fresh Pig Brains1 M GTP1 M Magnesium SulfatePM buffer =100 mM Pipes, pH 6.9?2 mM EGTA?1 mM Magnesium Sulfate2 mM DTTPM-4M Buffer =100 m
Tubulin－Basics
I. Useful Values1 mg/ml tubulin = 10 μM (assuming MW of ab-tubulin heterodimer is 100,000; in reality it is ~110,000 but almost all tubulin labs use t
Recycling－Tubulin
Recycling TubulinWe "recycle" tubulin fractions stored at -80?C after the PC column and store the recycled tubulin in small aliquots for day-to-day us
Preparation－of－tubulin
Although many protocols for tubulin preparation are available, the procedure described below is the simplest and highest yielding preparation I have d
Tubulin－Basics
I. Useful Values1 mg/ml tubulin = 10 μM (assuming MW of ab-tubulin heterodimer is 100,000; in reality it is ~110,000 but almost all tubulin labs use t
TUNEL－labeling
In Situ Cell Death (Apoptosis) Detection by TUNEL labelingby Boehringer Mannheim (Catalog No. 1684809), modified by Josiah N. Wilcox andJosé C. Rodrig
Biosynthetic－labeling
How long should cells be labeled??The ideal length of time to label cells depends on the protein of interest and the label that you are using. If you
Immunofluorescent－Localization－of－Tubulin
LEVEL IIMaterialsCoverslip cultures of an appropriate monolayer cell linePhosphate buffered saline (PBS)Acetone/Methanol (absolute) in a 50:50 volume
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 microtubules with a
Preparation－of－Segmented－and－Polarity－Marked－Microtubules
Preparation of Segmented and Polarity Marked Microtubules?Segmented and polarity-marked microtubules are very useful for many different types of?in vi
Detection－by－TUNEL－labeling
In Situ Cell Death (Apoptosis) Detection by TUNEL labelingby Boehringer Mannheim (Catalog No. 1684809), modified by Josiah N. Wilcox,José C. Rodriguez
CMFDA－Labeling－of－Platelet
OUTLINECMFDA (5-chloromethylfluorescein diacetate) is a lipophilic tracer that has an enormous advantage over ordinary tracers (e.g. FITC) because it
Immunofluorescence－Labeling－of－Cells
實驗概要Antibodies are an ?important tool for demonstrating both the presence and the subcellular ?localization of an antigen. Cell staining is a very ver
Arachidonic－Acid－Labeling
1) Grow cells to a density of 5-8 X 105 cells/ml in RPMI 1640 containing serum.2) Pellet cells and wash 1 time with room temperature PBS.3) Resuspend
BrdU－Labeling－Protocol
實驗概要The thymidine analog, 5-bromo-2-deoxyuridine (BrdU),is a common reagent used for cell proliferation assays and for the detection of apoptotic
Preparation－of－tubulin2
DAY 2: Cycling preparation of MT protein.Keep the brains in an evacuated plastic ziplock bag buried in ice from the time of slaughter during transport
porcine－brain－tubulin－prep
Although many protocols for tubulin preparation are available, the procedure described below is the simplest and highest yielding preparation I have d
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 phosph
DNA－labeling－by－nick－translation
DNA labeling by nick translationreagents:?DNA for labeling (concentration c > 150 ng/μl)?modified nucleotides:?Biotin-16-dUTP,?Digoxigenin-11-dUTP, co
SDS－Gel－Electrophoresis－of－Tubulin\MAPs
MaterialsStock Acrylamide: (30%T:0.8%C)30% by weight of acrylamide0.8% by weight of N,N'-bis-methylene acrylamideSeparation Gel (Final Concentrati
Tubulin－Polymerization－with－GTP/GMPCPP/Taxol
I. Solutions & SuppliesII. Prepolymerization ClarificationIII. GTP PolymerizationIV. Taxol PolymerizationV. GMPCPP PolymerizationVI. Determining Conce
Large－Scale－Tubulin－Preparation——2
III. Pouring a 1L Phosphocellulose (PC) ColumnResin: Whatman P11 Cellulose Phosphate -- fibrous cation exchanger(1 gram of PC swells to about 4 ml pac
Basic－Method－for－Indirect－Immunofluorescence－Labeling
Basic Method for?Indirect?Immunofluorescence LabelingBackgroundThis is the method for?indirect?immunofluorescence labeling; that is, the antibodies?do
Metabolic－Labeling－of－Cells－with－35S
1) Transfer to a 24 wells plate the desired colonies.2) Once the cells are attached (at least 8 hours after tripsinizing them) add ~1 ml ofDME (met-,
ThiolReactive－Probe－Labeling－Protocol
實驗概要Invitrogen ?offers several fluorescent and biotinylated phalloidin and phallacidin ?derivatives for labeling F-actin. These phallotoxins, isolated
細胞組分和細胞器——細胞器分離
Labeling Microtubules?(Molecular Dynamics Inc.??)Microtubules are involved in many aspects of cell motion including propulsion, mitosis, growth, and o
In－Situ－Cell－Death－(Apoptosis)－Detection－by－TUNEL－labeling
Protocol for Frozen Sections:Warm 150ml 4% Paraformaldehyde/1x PBS to RT. Fix slides in it, 20 min., RT.1x PBS rinse, 2 times.1x PBS, 30 min., RT. Beg