Application
Western blot
Sample
Mouse Cell lysate - whole cell (Mouse T-cell lymphoma and splenocytes)
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris)
Loading amount
40 µg
Specification
Mouse T-cell lymphoma and splenocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Other product details
Incubation time
18 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 3% BSA 2% Milk in PBST
Dilution
1/1000
Secondary antibody
Dilution
1/10000
Name
Non-Abcam antibody was used: HRP-conjugated Goat Anti-Rabbit
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Detection
Detection method
SuperSignal West Pico
Exposure
10 second(s)
Bands
Specific: 41 kDa Non-specific: 50-60 kDa
Positive control
EL4 T-cell lymphoma
Additional data
Additional Notes
Protein lysates from mouse EL4 T-lymphoma cells (Lane1) and whole splenocytes from C57BL/6 mice (Lane 2) were prepared using radioimmunoprecipitation assay buffer containing 1x protease inhibitor cocktail and 0.5 mM sodium orthonovanadate. Protein concentrations were determined and 40µg of each lysate was resolved on a 4-12% Bis-Tris gel and then transferred to nitrocellulose membrane. The membrane was blocked for 1 hour at room temperature with 5% non-fat milk in PBST. The blot was then probed with anti-CKII alpha prime at a dilution of 1:1000 in 3% bovine serum album in PBST with 2% non-fat milk at 4°C, overnight. HRP conjugated Goat anti-rabbit IgG was used at a dilution of 1:10000 in 3% bovine serum album in PBST with 2% non-fat milk, for 1 hour at room temperature. The HRP-conjugated secondary antibody was detected using West Pico Chemiluminescent Substrate and images acquired using a ChemiDOC XRS imaging system. Very few non-specific bands were detected. Exposure times may vary with the use of different secondary antibodies.
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.
Dr. Tomar Ghansah
Verified customer
提交于 Aug 21 2012