Application
Western blot
Loading amount
10 µg
Gel Running Conditions
Reduced Denaturing (5-20% gradient gel)
Sample
Human Cell lysate - whole cell (293T)
Specification
293T
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
Other product details
Incubation time
14 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 1% skim milk in PBST
Dilution
1/1000
Secondary antibody
Name
Non-Abcam antibody was used: HRP anti-Rabbit IgG (H+L)
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/5000
Detection
Detection method
ECL
Bands
Specific: ? kDa Non-specific: 40, 50, 60, 70, 75 kDa
Exposure
20 second(s)
Additional data
Additional Notes
1. Mock: Non-transfection control
2. Luc-2CP gene was cloned from Promega pGL series vector and inserted into the pHW72 vector. For detail, kindly see the reference Manzoor et al 2009 , 1572-1578. The luc2CP reporter gene contains hCL1 and hPEST, both of which are protein destabilization sequences.
3. ORF of Luc2CP was cloned in the pCAGGS vector (mammalian expression vector).
4. ORF of firefly luciferase was cloned in the pCAGGS vector. The luciferase protein does not contain protein destabilization sequences (hCL1 and hPEST).
Procedure 293T cells were transfected in 6-well plate with indicated plasmids.
After 48 hrs, growth media was removed, cells were suspended and washed twice with chilled PBS. Cell lysates were prepared in lysis buffer containing protease inhibitor (Complete mini).
Cell lysates were centrifuged at 14k for 12 minutes (4C).
Sample loading: Cell lysates were diluted 1:2 with laemmli buffer containing 2-mercaptoethanol. Samples were loaded @ of 20ul/well.
Abcam response
Thanks for your honest review of your experience using this antibody in western blotting. This is the first data we have regarding use of ab21176 in western blotting.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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Verified customer
提交于 Nov 12 2013