Lamond Lab 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