Download removes proteins

Molecular Biology Bio4751 Spring 2003
Gary A. Bulla, PhD
Methods in Molecular Biology
RNA quantitation
a. Northern
1. Lyse with detergent,
2. Phenol extract (removes proteins)
3. Precipitate RNA
4. Load onto agarose gel
5. Transfer RNA to nylon membrane
6. Add radioactive DNA or RNA to detect individual
species
Nylon
membrane
probed with
labeled a1antitrypsin
RNA, then
tubulin DNA
RNA quantitation
b. RNase protection
1. Lyse with detergent
2. Phenol extract (removes proteins)
3. Precipitate RNA
4. Incubate with radioactive antisense RNA
5. Degrade single strand RNA with RNases
6. Load on 8% PAGE
mRNA
AAAAA
Radioactive
antisense RNA
Hybridize
RNase treat
1.
2.
Heat , PAGE
Expose to film
RNA quantitation
c. Primer extension
RNA pol II holoenzyme
Histone/DNA ratios
TATAA
Prevent reinitiation
Primer extension analysis
mRNA
Primer
+dNTPs
Heat, PAGE, probe
Also used to identify locations where transcription starts
Molecular Biology Bio4751 Spring 2003
Gary A. Bulla, PhD
Western (protein detection)
A. Immunoblot
Cell extract
PAGE (+ SDS)
Incubate with anti-Rx
Transfer to nylon membrane
Alkaline
peroxidase
Rx
Anti-myc
AntiAnti-Rx
Enhancer
B. Radiolabeling
Transfect C ells
Western
35S
amino acid,
immunoprecipitate
electrophoresis
Genetically Modified Foods
Includes frost-resistant tomatoes
Disease-resistant sweet potatoes
Muscle-rich cattle
…..and many others
Last month• Zambia’s government rejected 1000s of tons of corn from
US because it may contain some GM kernels
•Approx 2.9 people at risk of starvation from droughtinduced famine since 2001
•35,000 will die by 2003 if food not provided (WHO)
•GM corn produces a bacterial toxin that is toxic to insects
•GM corn used world-wide for 6 years without adverse
effects (FDA)
How can we detect measure gene activation?
DNA
RNA
Northern
RNase Protection
Primer extension
Protein
Western
How do we examine DNA-protein interactions?
Mad
Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift)
DNaseI protection
Photo-crosslinking
How do we examine protein-protein interactions?
GST pull-down
EMSA Supershift
Co-immunoprecipitation
How can we measure promoter activity?
Answer- Link it to a gene that is easy to monitor
a1AT
-261
CAT
(Chloramphenicol acetyl transferase)
+44
Luciferase
B-galactosidase
CAT assay
a1AT
CAT
Lyse cells, mix with 14Cacetyl CoA, extract and
apply to thin layer
chromatography
Acetylated
Chloramphenicol
migration
Un-acetylated
Chloramphenicol
+ HNF1
- - + +
a1AT
-261
CAT
+44
- +
ControlTK-CAT
Protein-DNA interactions
EMSA (Gel Shift)
DNAseI footprinting
Photo-crosslinking
EMSA (Gel Shift assay)- 4% PAGE (non-denaturing)
-To detect protein-DNA interactions
- Usually transcription factor binding
Theory
80V, 3hr
15 min.
Expose to film
PAGE
Labeled DNA + protein
DNAprotein
complexes
Unbound DNA
fos jun
TRE
TATA
J=jun
F=fos
M=myc (another bZIP
protein)
C= bZIP domain only
Note- complexes
migrate according
to protein size
Fig. 12.31- Fos and jun binding to a TRE
Observe• jun or fos cannot bind alone (lanes 1-3)
• Jun+ fos does bind (lane 4)
• only bZIP domain (C) is required for binding (lanes5, 10 , 11)
• another bZIP factor (myc) fails to allow fos or jun to bind (lanes 14-15)
DNAseI footprinting
32P
Footprinting by DNaseI and Cu++
Experiment
TFIID, A and/or B added to DNA
treat with DNaseI or Cu++
polyacrylamide gel electrophoresis
(PAGE)
Observe•TFIID binds poorly
•A + D binds strongly
•B doesn’t enhance binding of D+A
Fig. 11.4 DNaseI footprinting
DNase I digestion
products
Photocrosslinking)
• To identify proteins which bind DNA
Fig. 6.25
Fig. 6.24
Photocrosslinking)
* * * * * * * *DNA
UV, nuclease
*
*
TFIID + P-32 labeled promoter DNA which
contains bromodoexyuridine (BrdU)
UV irradiate (causes BrdU to be crosslinked
to proteins it contacts)
nuclease
SDS-PAGE
Fig. 11.15- TAF 250 and 150 bind promoter DNA
Health stupidity reigns supreme
• Magnet therapy
• electronic ab exercisers- called “pump fiction” by
Federal Trade Commission -ftc/gov/opa/2002/05/projectabsurd.htm
• Acupuncture-No proven benefit in controlled studies
• Chiropractic medicine- only useful for lower back pain. Period.
All are largely accepted based upon “wart phenomenon”
To make a lie into a truth-
“Say it loud, say it often”
G. Gordon Liddy
How can we detect measure gene activation?
DNA
RNA
Northern
RNase Protection
Primer extension
Protein
Western
How do we examine DNA-protein interactions?
Mad
Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift)
DNaseI protection
Photo-crosslinking
How do we examine protein-protein interactions?
GST pull-down
EMSA Supershift
Co-immunoprecipitation
Protein-protein interactions
How do we determine identify protein-protein interactions?
Method- Epitope Tagging
Ligate a small peptide onto a protein, introduce that protein
into cells, then lyse the cells, and use antibodies raised
against the small peptide to bind the protein plus any
proteins interacting with that protein.
Gene of interest
ATG
TAA
ATG
TAA
Promoter
FLAG epitope
(7-9 amino acids)
Poly-Adenylation
sequence
Epitope-tagged protein
3A, slide 5
Method- Epitope Tagging
Figure 10.13
RNA polymerase II structure- yeast has 12 subunits
How do we determine identify protein-protein interactions?
HDAc
Co-immunoprecipitation
Example- Epitope tagging experiment
FLAG
Epitope-tagged histone deacetylase (HDAC2)
to generate FLAG-HDAC2
Introduce FLAG-HDAC2 + Mad1 plasmids into cells
Prepare cell extracts
Flag
Sin3A
Mad
HDAC
Ac
Immunoprecipitate
with anti-FLAG Ab
PAGE
Transfer to membrane
Probe with anti- SinA
or anti-Mad1
HDAC
Sin3A
Mad
Flag
Epitope tagging experiment results
Ac
Fig. 13.38- Evidence for ternary complex involving
HDAC2. Sin3A and Mad1
ObserveFLAG alone doesn’t interact with Sin3A or Mad1 (lanes 1-3)
HDAC2 interacts with Sin3A (Lane 4)
Mad1, but not mutant Mad1pro, interacts with Sin3A (lanes 5 and 6)
9E, slide 46
Another clever assay for protein-protein interaction
M2 High Sensitivity Capture Assay
CMV
FLAG
CBP
PolyA
CMV
C-myc
HNF1a
PolyA
Co-transfect Cos7 cells
Alkaline
peroxidase
Anti-myc
HNF1
CBP
Anti-FLAG
96-well format
How do we determine whether a protein is a histone acetyltransferase (HAT)?
Assay1. separate nuclear protein on SDS-PAGE
impregnated with histones
2. incubate gel with tritium -labeled AcCoA, wash away
All nuclear proteins
Ac*
Ac*
H4
H3
H2A
H2B
Ac
*
Ac
*
Fig. 13.33 Activity gel assay for HAT activity
How do we identify methylated DNA?
CCGG
HpaII
•Digest genomic DNA with
enzyme pair
•Load onto agarose gel
•Southern transfer
•probe with 32-P DNA
CCmGG
MspI
CCGG
DNA probe
Methylation analysis: The results of MspI and HpaII cleavage are
compared by Southern analysis
How does one find “open” vs “Closed” DNA
Inactive
DNase sensitivity assay
DNAseI
Active
DNAseI
Remove proteins
Remove proteins
Cut with restriction enzyme
6kb
4kb
6kb
4kb
3kb
5kb
5kb
3kb
Isolate chromatin
Treat with DNaseI
Remove protein,
Isolate DNA
Digest with BamHI
Agarose gel
Southern blot
Probe: a-globin
Ovalbumin
MSB= non-expressing cells
Fig. 13.31 Dnase I hypersensitivity of an active gene
Transcription run-off assay
• To monitor transcriptional activity of a gene
Measure transcription directly. Thus post-transcriptional
processing in not a concern
Figure 5.27
8B, slide 18
Transcription run-off assay
SP1
375 nt transcript
TBP
TATAA
Note- Each lane contains RNA pol II
+ TFIIA,B,E and F
bh= bacterially derived human TBP
vh=virus derived human TBP
Fig. 11.18- TBP alone can’t
respond to Sp1