Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa)
Gel Running Conditions
Reduced Denaturing (8% gel)
Loading amount
25000 cells
Treatment
2,5mM Thymidine or 50 ng/ml nocodazole for 16hrs and released as indicated
Specification
HeLa
Blocking step
2%milk, 1%BSA in PBS-Tween-20 (0,1%) as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: 20°C
Other product details
Incubation time
2 hour(s) and 0 minute(s) · Temperature: 20°C · Diluent: 25% blocking buffer in PBS-Tween-20
Dilution
1/500
Secondary antibody
Dilution
1/5000
Name
Non-Abcam antibody was used: DAKO
Host species: Rabbit
Clonality: Monoclonal
Conjugation: Horse Radish Peroxidase
Host species: Rabbit
Clonality: Monoclonal
Conjugation: Horse Radish Peroxidase
Detection
Detection method
ECL+
Exposure
30 second(s)
Bands
Specific: 71 kDa Non-specific: many (with long expo kDa
Additional data
Additional Notes
HeLa cells syncronized with O/N Thymidine block or nocodazole block and released as indicated:
Lane 1: untreated
Lane 2: 0 hrs release after thymidine block (G1)
Lane 3: 4 hrs release after thymidine block (S)
Lane 4: 8 hrs release after thymidine block (G2/M)
Lane 5: 0 hrs release after nocodazole block (M)
Although Dyneine gives the dominant signal, longer exposures give a lot of background bands
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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投稿 Dec 15 2010