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)
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.
Covalently conjugate antibodies to beads for affinity
purification
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)
Gels/blots