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Friday, December 12, 2008

Biochemical Protocol 2. Microscope Technology (2)

Image capture
Image analysis

Image Galleries (Links to collections of beautiful images that use a number of microscopic imaging techniques0

Introduction

Introduction (A look at the basics of what makes up a digital image, color and file formats, as well as a few links on human vision)

Laser microscopy

Laser microscopy: stereo-viewer

Light & Microscopes Larry Legg's funky introduction

Light Microscopy (Links to excellent resources that introduce concepts of light & optics, as well as how light microscopes work)

Light microscopy

McCrone Research Institute Teaching light microscopy, pollen and spore identification

Measurement of Cell Organelles

Measurements: Ocular and Stage Micrometers

Measuring Area

Measuring Depth

Measuring Volume

Microscopy - Wikipedia, the free encyclopedia

Microscopy Resources (General information about professional societies, vendors, web resources and email discussion groups)

Microscopy-UK general resource, training, and supplies

Phase Contrast illumination and other methods, Larry Legg

Polarized light illumination - Do-it-yourself, cheap

Polarized Light illumination, David Walker

Preparation in TEM

Sample Preparation of Confocal Microscopy

Software (Links to resources for several commercial and freeware image editing programs)

Stereo vision

The Bright Field Microscope

The Inverted Phase Microscope

The Phase Contrast Microscope

The Transmission Electron Microscope

Use of Darkfield Illumination

Use of Oil Immersion (100x)

Video microscopy (Microscopy & Imaging Resources on the WWW)

Biochemical Protocol 3. Histochemistry Protocols

Alkaline Phosphatase Localization

Autoradiography

Basophilia

Cell fixation - room temperature paraformaldehyde (good for paraspeckles/speckles for 5 min; most other things for 10 min)
Cell fixation - methanol

Conjugating antibodies to fluorescent probes

Histology Protocol

Immunoflourescence of MEFs
Mouse Necropsy
Necropsy form
Paraffin embedding and Sectioning
Staining Protocols
H & E staining
BrdU Staining Histological Sections
X-gal Staining
HRP-DAB Immunostaining
AP-BCIP/NBT Immunostaining
GFP & BrdU In Situ Immunoflourescence
Fixation, permeabilization and immunostaining of cells
(contains images and movies)
Cell fixation - warm paraformaldehyde (preserves microtubules)

Immunofluorescent Localization of Tubulin

Methyl Green-Pyronin Staining of DNA and RNA

Periodic Acid Schiff (PAS) Reaction

Selective Staining: Prepared Slides

The Feulgen Procedure for DNA

Biochemical Protocol 4. Protein

Affinity column preparation and Immunoprecipitation

Affinity purification of Antibodies

B-PER Bacterial Protein extraction reagent (Pierce)

Bradford quantification of protein concentration

Crosslinking Protocol

Crosslinking the antibody to the beads protocol | Abcam

Factor Xa Cleavage of GST fusions

GST-fusion protein injection into rabbits

GST-protein fusion expression, purification and other uses

His-fusion protein purification (Novagen/Qiagen)

Histone Kinase Assay
Western blot
p27 ELISA

Immunoblot Anaylsis

Immunoprecipitation

Protein Interactions Captured by Chemical Cross-linking: One-Step Cross Linking.

Protein precipitations

Protein preparation (from plants tissue)

Protein staining

Protocol: Cross-linking of IgG to Protein A or G Beads: NEB

Radio-labeling of protoplasts

SDS/Native PAGE

Silver staining

Stripping blots to reprobe with different antibodies

UV Crosslinking Protocol

Western blots

Biochemical Protocol 5. Cell Fractionation

Cell Fractionation Protocol.
Chromatin isolation
Spliceosome purification

Cell Fractionation Protocol.
Covalently conjugate antibodies to beads for affinity purification
Nucleolar isolation
(contains images and movies)
Cajal body isolation
(contains images and movies)
Lysing cells for release of both cytoplasmic and nuclear proteins

Equilibrium Density Gradient - Percoll

Low Speed Separaton of Cells

Preparation of nuclear extract from plated cells

Sedimentation Densities

Separation of Cells by Velocity Sedimentation

Sucrose Fractionation of Castor Bean

Biochemical Protocol.6. Enzymology

Addition of enzyme inhibitors

Advanced analysis of enzyme parameters

Computer simulation

Determination of Km and Vmax

Effects of ph

Effects of temperature

Enzyme Assay22-28

Enzyme Assay36-42

Enzyme Assay43-47

Enzyme concentration

Extraction of tyrosinase

Fucosyltransferase assay

Introduction

Kinetic analysis

Phosphatase assay

Preparation of standard curve

Protein concentration/enzyme activity

Biochemical Protocol.7. RNA

Amino-allyl labeling

BioAnalyzer
TRIzol plus RNEasy Mini
RNEasy Midi
TRIzol product insert
RNA amplification
RNA gel electrophoresis

C. elegans RNA prep: Michael Koelle

In vitro transcription reaction

Isolation of poly-A RNA

LiCl RNA Preparation: MGH

Northern blots

Northern Blots: Michael Koelle

Nucleotide composition of RNA

Orcinol determination of RNA

Phenol extraction of rRNA (rat liver)

RNA and protein extraction from the same sample

RNA prepararion (from plants tissue)

RNA Preparation: MGH

Spectrophotometric analysis of RNA

Sucrose density fractionation

Worm RNA Preparation: MGH

Biochemical Protocol.8. DNA (1)

ABC Random Amplification
Alcohol Precipitation of DNA
AML sample DNA preparation

Agarose gel electrophoresis

Agarose gel electrophoresis

Boomerang DNA Amplification Single primer DNA amplification

cDNA amplification from l-phage library

cDNA library screening

Characterization of DNA

DIG DNA labelling and hybrid detection (Boehringer Mannheim)

Direct Automated Sequencing off Bacterial Genomic DNABruce Rhoe

DNA extraction from agarose (Qiagen)

DNA - dische diphenylamine determination

DNA Fragment Isolation from LM agarose

DNA Ligation

DNA detection
DAPI chromosome identification

DNA preparation by cryostom tissue dissection
DNA labeling by nick translation

Ethanol precipitation of DNA

Extraction of DNA from bovine spleen

Fluorometry
FISH