Application
Western blot
Sample
Human Cell lysate - whole cell (MCF7 breast carcinoma cell line)
Loading amount
30 µg
Specification
MCF7 breast carcinoma cell line
Treatment
DMSO or CAN508 for 24hr
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
Other product details
Dilution
1/10000
Incubation time
16 hour(s) and 0 minute(s)
Secondary antibody
Name
Non-Abcam antibody was used: rabbit anti-mouse IgG, peroxidase-conjugated
Host species: Rabbit
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Rabbit
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Detection
Detection method
ECL+
Exposure
5 minute(s) and 0 second(s)
Bands
Specific: 220-240 kDa kDa
Positive control
MCF7 cells treated with different concentrations of CAN508 (a novel CDK9 selective inhibitor) for 24 h showed dose-dependent decrease of Ser 5 phosphorylation, indicating CDK7/9 inhibition in cells.
Negative control
MCF7 cells treated with 0.1% DMSO for 24hr. No decrease of the signal was observed when compared to untreated cells.
Additional data
PubMed ID
17064068: View
Additional Notes
For direct immunoblotting, total cellular protein lysates were prepared by harvesting treated cells in Laemmli sample buffer. Proteins were separated on 10% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membrane. The blotted membranes were stained with Ponceau-S in 1% aq AcOH to verify equal protein loading, destained, and blocked in PBS and 0.1% Tween 20 (PBS-T) with 5% low fat milk. The membranes were then incubated with specific antibodies overnight. After washing three times in PBS-T, the membranes were incubated with a 1:2000 dilution of peroxidase-conjugated secondary antibodies. After another three washes in PBS-T, peroxidase activity was detected using ECL+ reagents.
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提交于 Jan 02 2007