detecting endogenous macromolecules detecting endogenous structures, cell marking, small molecules...
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Detecting Endogenous Macromolecules
Detecting Endogenous structures ,cell marking, small molecules
Detecting ‘Planted’ Reporters
Detecting Endogenous MacromoleculesProteinNucleic acids (RNA, DNA)
Detecting Endogenous structures ,cell marking, small molecules
Detecting ‘Planted’ Reporters
GREEN marks theposition of the protein fibronectin in this frog embryo
Section
HOW DO WE DO THIS?
RED marks nuclei
MakingPolyclonals
serum + or - purif.
MCB 6.2(‘0601’)
Monoclonals
PAGE PolyAcrylimide Gel Elecrophoresis
MCB 3
Western or Immunoblot MCB 3.5 “3EIMMBLOT”
Immunocytochemistry
Most common enzyme conjugates:
Alkaline phosphataseHorseradish Peroxidase
Fluorescent microscopy
FITC secondary
Anti-fibronectinThen FITC
Fluorescence, rather than a converted substrate, as secondary to
mark protein’s presence
RED, PI, nuclear
counterstain
Principle of Confocal microscopy
Confocal – What it offers
Regular ConfocalFluorescence microscopy
Actin Gurken
Hunchback Kruppel
Immunogold
Immunogold
SO:
Immunofluorescence
Immunocytochemistry
For protein: antibody-antigen
For nucleic acid: n.a. complementarity
Tracking specific macromolecules
MCB 7.2PCR
Start here week2/3
In situ hybridization with 35S RNA probes1 2 3 4
In situ hybridization using radioactive probe- expose photographic emulsion
In situ –Shh
FGF8 in situ
FISH
Northern
Northern or SLOT-BLOT
Detecting Endogenous MacromoleculesProteinNucleic acids (RNA, DNA)
Detecting Endogenous structures ,cell marking, small molecules
Detecting ‘Planted’ Reporters
Detection endogenous RNAs (hybridization + . . .)
Northern - or dot/slot blotDevelopmental Northern or SLOT-BLOT
In Situ Hybridization
MicroarrayTiling Microarray
RNA seq.Single cell RNA seq.
RIBOSOME PROFILING- on way to proteome
MICROARRAY ANALYSIS
Array analysis: see animation from Griffiths
Figure 4.16(1) Microarray Analysis of Those Genes Whose Expression in the Early Xenopus Embryo Is Caused by the Activin-Like Protein Nodal-Related 1
(Xnr1)
Figure 4.16(2) Microarray Analysis of Those Genes Whose Expression in the Early Xenopus Embryo Is Caused by the Activin-Like Protein Nodal-Related 1
(Xnr1)
Figure 4.15(1) Microarray Technique
Figure 4.15(2) Microarray Technique
Detection endogenous RNAs (hybridization + . . .)
Northern - or dot/slot blotDevelopmental Northern or SLOT-BLOT
In Situ Hybridization
MicroarrayTiling Microarray
RNA seq.Single cell RNA seq.
RIBOSOME PROFILING- on way to proteome
NGS (Next Generation Sequencing)
Detecting Endogenous Macromolecules
Detecting Endogenous structures ,cell marking, small molecules
Detecting ‘Planted’ Reporters
Other markers of cells ‘Staining’ cells to follow cells / lineagesMarkers for small moleculesMarkers for cell compartments
Vital dye injection into cells to follow cell lineage
Flourecent dye injection into cells to follow cell lineage
Dye injection into cells to map neurites (here, axons from retinal neurons to tectum)
Other markers of cells ‘Staining’ cells to follow cells / lineagesMarkers for small moleculesMarkers for cell compartments
Hoescht-Dye (or DAPI) Antibody, FITC to P granules
A dye that fluoresces when it binds Ca++ Time series
Detecting Endogenous Macromolecules
Detecting Endogenous structures ,cell marking, small molecules
Detecting ‘Planted’ Reporters To see protein, (OR RNA)
MCB 5.1
ReporterConstructs
Myf-5 Driven Beta-gal
X-gal
Retinal-specific gene’s promoter driving GFP
Acrosin-GFP
GFP spindleshttp://www.duke.edu/web/microlabs/endow/moviepage.html
What about seeing RNA molecules in cell
