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Detecting Endogenous Macromolecules
Detecting Endogenous structures,
cell marking, small molecules
Detecting ‘Planted’ Reporters
Detecting Endogenous Macromolecules
Protein
Nucleic acids (RNA, DNA)
Detecting Endogenous structures,
cell marking, small molecules
Detecting ‘Planted’ Reporters
GREEN marks the
position of the
protein
fibronectin in
this frog embryo
Section
HOW DO WE
DO THIS?
RED marks nuclei
Making
Polyclonals
serum
+ or - purif.
MCB 6.2
Monoclonals
(‘0601’)
PAGE
PolyAcrylimide Gel Elecrophoresis
MCB 3
Western or Immunoblot
MCB 3.5 “3EIMMBLOT”
Immunocytochemistry
Most common
enzyme conjugates:
Alkaline phosphatase
Horseradish Peroxidase
FITC secondary
Fluorescent microscopy
Anti-fibronectin
Then 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
Fluorescence microscopy
Regular
Confocal
Actin
Gurken
Hunchback
Kruppel
Immunogold
SO:
Immunogold
Immunofluorescence
Immunocytochemistry
Tracking specific macromolecules
For protein: antibody-antigen
For nucleic acid: n.a. complementarity
MCB 7.2
PCR
Start here week2/3
In situ hybridization with 35S RNA probes
1
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 Macromolecules
Protein
Nucleic acids (RNA, DNA)
Detecting Endogenous structures,
cell marking, small molecules
Detecting ‘Planted’ Reporters
Detection endogenous RNAs (hybridization + . . .)
Northern - or dot/slot blot
Developmental Northern or SLOT-BLOT
In Situ Hybridization
Microarray
Tiling 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 blot
Developmental Northern or SLOT-BLOT
In Situ Hybridization
Microarray
Tiling Microarray
NGS (Next
Generation Sequencing)
RNA seq.
Single cell RNA seq.
RIBOSOME PROFILING- on way to proteome
Detecting Endogenous Macromolecules
Detecting Endogenous structures,
cell marking, small molecules
Detecting ‘Planted’ Reporters
Other markers of cells
‘Staining’ cells to follow cells /
lineages
Markers for small molecules
Markers 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 /
lineages
Markers for small molecules
Markers 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
Reporter
Constructs
Myf-5 Driven Beta-gal
X-gal
Retinal-specific gene’s promoter driving GFP
Acrosin-GFP
GFP spindles
http://www.duke.edu/web/microlabs/endow/moviepage.html
What about seeing
RNA molecules in cell