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Incubate at 37C for 45 min and then stop reaction with 1L of
0.5 M EDTA then add 15 L of DEPC treated water.
Method used in labeling nucleoti
des probe
End labeling
Polymerase-based labeling
Nick translation of DNA
ATP
End-labeling probe DNA
P
Component for loading sample
Gel shift binding buffer 2 L
Nuclear extract 50 g/ reaction
Labeled probe 1 L
Nuclease free water up to 10 L
Treatments CPM/L
Blank 47.8
59.3
Reference 14,586,732.2
Before Chroma spin column 515,764.3
After Chroma spin column
188,179.4
Labeled 32P
Oligonucleotide 1 2 3 4 5 6
Origin
1 = SP1
2 = AP1
3 = TFIID
4 = NFkB
5 = Oct
6 = CREB
Free probe
No competitor Competitor
Nuclear protein,
50 mg
Protein binds to
unlabelled
competitor
Gel electrophoresis
Free probe
Autoradiography
Competiters
(cold probe)
KB-V1
P3P4
Curcumin treated cells (KB-V1)
Cont SP1 AP1 AP2 Oct CREB NFkB TFIID Cont SP1 AP1 AP2 Oct CREB NFkB TFIID
CREB consensus sequence
Cold CREB Cold probe 50 molars excess
Competitors
- + Cont SP1 AP1 AP2 TFIID NFkB Oct CREB
Free probe
P3P4
1 2 3 4
Origin
Free probe
C 2p B C C Sig Bk
Origin
Note
C = Control
2p = Isopropanol step
B = Bisdemethoxycurcumin
Sig = Sigma
Bk = Bisdemethoxycurcumin
from Kalsec
No supershift Supershift
Ab
Nuclear protein,
50 mg
Protein binds to
unlabelled
competitor
Gel electrophoresis
Supershift
Free probe
Autoradiography
Supershift
Component for loading sample for Supershift assay
Anti-CREB 2 g
Gel shift binding buffer 2 L
Nuclear extract 50 g/ reaction
Labeled probe 1 L
Nuclease free water up to 10 L
1 2 3 4 5
6 7 Origin
Free probe
Supershift
1 = control
2 = NE+Anti-CREB