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Detecting Endogenous Macromolecules Detecting Endogenous structures, cell marking, small molecules Detecting ‘Planted’ Reporters.

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Presentation on theme: "Detecting Endogenous Macromolecules Detecting Endogenous structures, cell marking, small molecules Detecting ‘Planted’ Reporters."— Presentation transcript:

1 Detecting Endogenous Macromolecules Detecting Endogenous structures, cell marking, small molecules Detecting ‘Planted’ Reporters

2 Detecting Endogenous Macromolecules Protein Nucleic acids (RNA, DNA) Detecting Endogenous structures, cell marking, small molecules Detecting ‘Planted’ Reporters

3 GREEN marks the position of the protein fibronectin in this frog embryo Section HOW DO WE DO THIS? RED marks nuclei

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5 Making Polyclonals serum + or - purif.

6 MCB 6.2 (‘0601’) Monoclonals

7 PAGE PolyAcrylimide Gel Elecrophoresis MCB 3 Western or Immunoblot MCB 3.5 “3EIMMBLOT”

8 Immunocytochemistry Most common enzyme conjugates: Alkaline phosphatase Horseradish Peroxidase

9 Fluorescent microscopy FITC secondary

10 Anti-fibronectin Then FITC Fluorescence, rather than a converted substrate, as secondary to mark protein’s presence RED, PI, nuclear counterstain

11 Principle of Confocal microscopy

12 Confocal – What it offers Regular Confocal Fluorescence microscopy

13 Actin Gurken

14 Hunchback Kruppel

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17 Immunogold SO: Immunofluorescence Immunocytochemistry

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22 For protein: antibody-antigen For nucleic acid: n.a. complementarity Tracking specific macromolecules

23 MCB 7.2 PCR Start here week2/3

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28 In situ hybridization with 35 S RNA probes

29 In situ hybridization using radioactive probe -expose photographic emulsion

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32 In situ –Shh

33 FGF8 in situ

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35 FISH

36 Northern Northern or SLOT-BLOT

37 Detecting Endogenous Macromolecules Protein Nucleic acids (RNA, DNA) Detecting Endogenous structures, cell marking, small molecules Detecting ‘Planted’ Reporters

38 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

39 MICROARRAY ANALYSIS

40 Array analysis: see animation from Griffiths

41 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)

42 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)

43 Figure 4.15(1) Microarray Technique

44 Figure 4.15(2) Microarray Technique

45 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 NGS (Next Generation Sequencing)

46 Detecting Endogenous Macromolecules Detecting Endogenous structures, cell marking, small molecules Detecting ‘Planted’ Reporters

47 Other markers of cells ‘Staining’ cells to follow cells / lineages Markers for small molecules Markers for cell compartments

48 Vital dye injection into cells to follow cell lineage

49 Flourecent dye injection into cells to follow cell lineage

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51 Dye injection into cells to map neurites (here, axons from retinal neurons to tectum)

52 Other markers of cells ‘Staining’ cells to follow cells / lineages Markers for small molecules Markers for cell compartments

53 Hoescht-Dye (or DAPI) Antibody, FITC to P granules

54 A dye that fluoresces when it binds Ca++ Time series

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56 Detecting Endogenous Macromolecules Detecting Endogenous structures, cell marking, small molecules Detecting ‘Planted’ Reporters To see protein, (OR RNA)

57 MCB 5.1 Reporter Constructs

58 Myf-5 Driven Beta-gal X-gal

59 Retinal-specific gene’s promoter driving GFP

60 Acrosin-GFP

61 GFP spindles

62 What about seeing RNA molecules in cell

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