Application
Western blot
Loading amount
22000 cells
Gel Running Conditions
Reduced Denaturing (NuPAGE® Novex® 4-12% Bis-Tris Gel,1.0 mm)
Sample
Human Cell lysate - whole cell (HeLa cells)
Specification
HeLa cells
Treatment
different siRNAs for 72h
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Other product details
Incubation time
18 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% BSA in PBST 0.1% plus 0.02% NaAzide
Dilution
1/500
Secondary antibody
Name
Non-Abcam antibody was used: IRDye 680 donkey anti rabbit
Host species: Donkey
Clonality: Monoclonal
Conjugation: IRDye® 680
Host species: Donkey
Clonality: Monoclonal
Conjugation: IRDye® 680
Dilution
1/10000
Detection
Detection method
LICOR
Bands
Specific: 270 kDa Non-specific: 105 kDa
Exposure
5 minute(s) and 0 second(s)
Negative control
on Target NonTargeting siRNA pool (1 in image)
anti beta-tubulin (with secondary antibody IRDye 800CW donkey anti mouse) as loading control
Positive control
A single siRNA and a siRNA pool against Sec16A were used on HeLa cells. Nontargeting siRNA pool was used as negative control.
Protein extraction 72h after transfection using 1x Sample Buffer. Novex® Sharp Pre-stained Protein Standard was used as marker.
Beta-tubulin was co-stained as loading control.
Strongest band at around 270 kDa is specific, as it is disappearing under siSec16A condition using two different siRNAs (2 &3 in image).
Additional data
Additional Notes
Image:
1: siNonTargeting pool onTarget
2: siC9Orf140 siGenome pool
3: siC9Orf140 sionTarget pool
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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提交于 May 27 2013