Title: Analyses of mRNA, S1 mapping and primer extension' Analyses of gene expression at the mRNA level: in
1Antibodies
- Analytical Techniques
- Utilizing Antibodies
- flow cytometry
- gel electrophoresis
- immunoprecipitation (IP)
- immunoblotting
- microscopy
- immunofluorescence (IFA)
- electron microscopy
- ELISA
- antibodies bind proteins with high specificity
and affinity - affinity chromatography
- analytical techniques
2Antibodies can be generated against protein
epitopes
3Antibodies can bind proteins with very high
affinities
4Polyclonal and monoclonal antibodies
5Indirect Immunocytochemistry
6The Enzyme-Lined Immunosorbent Assay (ELISA)
7Conventional ELISA
- bind protein to membrane or 96-well microplate
- neg. (and pos.) controls
- purity?
- incubate with 1o and 2o antibodies
- use soluble chromogenic substrates in 96-well
plates - quantify Ag or Ab
8- measure absorbance with ELISA microplate reader
9Screening Expression Libraries
- prepare library in expression vector
- cDNA better than gDNA
- 6X possible reading frames
- screen with protein based probe
- antibody/western blot
- ligand binding or other activity
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11Images of Conventional (A) vs Confocal (B)
Fluorescence (Drosophila embryo stained w
fluorescent probe against actin)
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13Specific markers for specific cells
14- METHODS FOR SHOWING INTERACTIONS
- 1. Between protein and DNA
- EMSA (electrophoretic mobility shift assay), gel
shift - In cell cultures transcription factor and
promotor - interaction
- 2. Between protein and another protein
- immunoprecipitation (in vivo)
- GST pull-down assay (in vitro)
- yeast two-hybrid system (in yeasto)
- Resonance energy transfers (FRET, BRET)
15EMSA
EMSA
DNA protein
control oligos
16EMSA
17EMSA
EMSA
DNA protein
antibody
18EMSA
Jun, Fos (AP-1) and MBF1 are needed to form a
complex with DNA
19METHODS FOR SHOWING INTERACTIONS IN
CELLS Between transcription factors and target
genes Between transcription factors and their
coactivators/repressors Between nuclear
receptors and their ligands
20In cell cultures
How to measure gene activation in eukaryotic
cells ?
Transfection assays
21In cell cultures
Coding region
Coding region
22In cell cultures
Coding region
Coding region
23In cell cultures
promotor-Firefly luciferase
standard-Renilla luciferase
plasmids
Cell culture - transfection
30-48 hours
24In cell cultures
promotor-Firefly luciferase
standard-Renilla luciferase
plasmids
luciferase
luciferase
luciferase
luciferase
luciferase
luciferase
25METHODS FOR SHOWING INTERACTIONS
- 2. Between protein and another protein
- immunoprecipitation (in vivo)
- GST pull-down assay (in vitro)
- yeast two-hybrid system (in yeasto)
- Resonance energy transfers (FRET, BRET) cell
- cultures, animals
26Immunoprecipitation
- affinity purification based on isolation of Ag-Ab
complexes - analyze by gel electrophoresis
- initially based on centrifugation of large
supramolecular complexes - high and ?equal amounts
- isolation of Ag-Ab complexes
- fixed S. aureus
- protein A-agarose
- protein G-agarose
- Bacterial proteins that bind IgG (Fc)
- protein A (Staphylococcus aureus)
- protein G (Streptococcus)
- binds more species and subclasses
27Typical IP Protocol
- 1. Solubilize antigen
- usually non-denaturing
- SDS excess of TX100
- 2. Mix extract and Ab
- 3. Add protein G-agarose, etc
- 4. Extensively wash
- 5. Elute with sample buffer
- 6. SDS-PAGE
- 7. Detection
- protein stain
- radioactivity
G
28- increase stability
- affinity purification
- detection/assay
- spectrophotometric
- binding assays
- antibodies
- export signals
Fusion Proteins
29Fusion Proteins
luciferase
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31Yeast 2-Hybrid Assay
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33Fluorescence Microscope
34Stain for DNA.
What is fluorescence?
Energy of light is inversely proportional to the
wavelength. For example, violet light has higher
energy than red light. Fluorescent molecules are
special molecules that absorb incident light of a
particular wavelength then emit light of a longer
wavelength. The absorption of light causes
electrons in the molecule to become highly
energetic. When the highly energetic electrons
return to their original state, they emit light.
Wave length of incident light must be shorter
than the wavelength of emitted light since some
energy is unavoidably lost as heat.
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36FRET
37FRET
38FRET
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40BRET
FRET
luciferase
BRET
luciferase
A
B
C
D
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