Protocols

SILAC-based Quantitative Proteomics


For details on sourcing reagents and preparing the media, download our SILAC Reagent Protocol.

For details on covalently coupling antibodies to Protein G sepharose, download our Covalent Coupling Protocol.

For details on cell fractionation and preparation of whole cell vs. nuclear and cytoplasmic lysates, download our Cell Fractionation Protocol.

For details on trypsin digestion of gel slices for MS analysis, download our In Gel Digestion Protocol.

Cell staining & microscopy


For details on working on with transiently transfected and stably expressed GFP-tagged proteins (including sections on live cell imaging, FRAP and FRET by acceptor photobleaching and FLIM/FRET), download our recent Methods in Molecular Biology chapter here.

Fixation, permeabilization and immunostaining of cells
(contains images and movies)

Cell fixation - warm paraformaldehyde (preserves microtubules)

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

Preparing proteins/antibodies/DNA/RNA for microinjection

CFP/YFP FRET measurement by sensitized emission

In vivo transcription

BrUTP labeling of RNA transcripts

Purifying nuclear structures


Nucleolar isolation
(contains images and movies)

Cajal body isolation
(contains images and movies)

For details on cell fractionation and preparation of whole cell vs. nuclear and cytoplasmic lysates, download our Cell Fractionation Protocol.

Chromatin isolation

Spliceosome purification

Covalently conjugate antibodies to beads for affinity purification

Tissue culture


Establishing stable cell lines

Fusing cells to create heterokaryons

Effectene (QIAGEN) tranfection of adherent mammalian cells (works well for HeLas and many other cell types)

Fugene (Roche) tranfection of adherent mammalian cellsDOTAP (Roche) tranfection of adherent mammalian cells

Calcium Phosphate tranfection of adherent mammalian cells (works well for 293 cells)

In vitro assays

Splicing assay

Phosphatase assay

Bacterial techniques


Release of recombinant proteins from bacterial cells using Novagen's Bugbuster

Purification of recombinant hexahistidine-tagged proteins

Purification of recombinant GST-tagged proteins

Gels/blots


Lysing cells for release of both cytoplasmic and nuclear proteins

Preparation of nuclear extract from plated cells

Stripping blots to reprobe with different antibodies

Silver staining

Food (favorite lab recipes collected over the years)


Quarkbrotchen

Zupfkuchen

Susan Veitch's World Famous Lemon Poppyseed Cake

Archa's Jaffa Cake

*recipes should only be prepared by competent chefs in a kitchen environment.




 

 


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