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