Analyses of mRNA, S1 mapping and primer extension' Analyses of gene expression at the mRNA level: in - PowerPoint PPT Presentation

1 / 41
About This Presentation
Title:

Analyses of mRNA, S1 mapping and primer extension' Analyses of gene expression at the mRNA level: in

Description:

Antibodies can be generated against protein epitopes ... standard-Renilla luciferase. promotor-Firefly luciferase. 2. Between protein and another protein ... – PowerPoint PPT presentation

Number of Views:1519
Avg rating:3.0/5.0
Slides: 42
Provided by: davidd125
Category:

less

Transcript and Presenter's Notes

Title: Analyses of mRNA, S1 mapping and primer extension' Analyses of gene expression at the mRNA level: in


1
Antibodies
  • 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

2
Antibodies can be generated against protein
epitopes
3
Antibodies can bind proteins with very high
affinities
4
Polyclonal and monoclonal antibodies
5
Indirect Immunocytochemistry
6
The Enzyme-Lined Immunosorbent Assay (ELISA)
7
Conventional 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

9
Screening 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

10
(No Transcript)
11
Images of Conventional (A) vs Confocal (B)
Fluorescence (Drosophila embryo stained w
fluorescent probe against actin)
12
(No Transcript)
13
Specific 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)

15
EMSA
EMSA
DNA protein
control oligos
16
EMSA
17
EMSA
EMSA
DNA protein
antibody
18
EMSA
Jun, Fos (AP-1) and MBF1 are needed to form a
complex with DNA
19
METHODS FOR SHOWING INTERACTIONS IN
CELLS Between transcription factors and target
genes Between transcription factors and their
coactivators/repressors Between nuclear
receptors and their ligands
20
In cell cultures
How to measure gene activation in eukaryotic
cells ?
Transfection assays
21
In cell cultures
Coding region
Coding region
22
In cell cultures
Coding region
Coding region
23
In cell cultures
promotor-Firefly luciferase
standard-Renilla luciferase
plasmids
Cell culture - transfection
30-48 hours
24
In cell cultures
promotor-Firefly luciferase
standard-Renilla luciferase
plasmids
luciferase
luciferase
luciferase
luciferase
luciferase
luciferase
25
METHODS 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

26
Immunoprecipitation
  • 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

27
Typical 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
29
Fusion Proteins
luciferase
30
(No Transcript)
31
Yeast 2-Hybrid Assay
32
(No Transcript)
33
Fluorescence Microscope
34
Stain 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.
35
(No Transcript)
36
FRET
37
FRET
38
FRET
39
(No Transcript)
40
BRET
FRET
luciferase
BRET
luciferase
A
B
C
D
41
(No Transcript)
Write a Comment
User Comments (0)
About PowerShow.com