Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa cells)
Gel Running Conditions
Reduced Denaturing (handcast 10% tris-glycine, 15 well)
Loading amount
40000 cells
Treatment
SENP1 siRNA treated to show specificity
Specification
HeLa cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Other product details
Incubation time
15 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: O/N incubation with light agitation
Dilution
1/1000
Secondary antibody
Dilution
1/5000
Name
Non-Abcam antibody was used: anti-rabbit 800
Host species: Rabbit
Clonality: Polyclonal
Conjugation: IRDye 800CW
Host species: Rabbit
Clonality: Polyclonal
Conjugation: IRDye 800CW
Detection
Detection method
Odyssey
Negative control
HeLa WCL from siRNA knocked-down SENP1 cells (lane 3)
Exposure
1 minute(s) and 0 second(s)
Bands
Specific: ~70 kDa Non-specific: 130 kDa
Positive control
HeLa whole cell lysate (WCL) (lane 2)
Additional data
Additional Notes
HeLa cells were harvested in 100 ´L 2x SB with 10% BME. Sonicated 3x 15 sec each, in a water sonicator. Samples were heated for 5 min at 95 ¯C, cooled, spun and loaded onto a freshly poured 10% Tris-Glycine gel. Gels were run at 110 V for 1 hour and 15min. Transferred gels using the Bio-Rad TransBlot Turbo (2.5 A, 25 V, 10 min). Gel lane descriptions: 1) MWM 2) WT HeLa WCL 3) siSENP1 KD HeLa WCL 4) GFP-SENP1 overexpression
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投稿 Jun 14 2017