Western blot abreview for Anti-p21 antibody [EPR362]

Average
Abreviews
Application
Western blot
Loading amount
15 µg
Gel Running Conditions
Reduced Denaturing (13%)
Sample
Human Cell lysate - whole cell (HeLa, HCT116 and MEF cell lysates)
Specification
HeLa, HCT116 and MEF cell lysates
Treatment
DMSO control (Ctl) and 7,5 ´M CPT-11 (CPT) for 24 hrs
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Other product details

Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 2% Milk in PBS-T buffer
Dilution
1/2500

Secondary antibody

Name
Dilution
1/10000

Detection

Detection method
ECL
Bands
Specific: 18 kDa Non-specific: 20 kDa
Exposure
10 second(s)
Negative control
HCT116 p21-/- cells

Additional data

Additional Notes
The antibody detects p21 after short exposure times. We used HeLa cell cycle lysates from Abcam (ab136811) to check for cell cycle dependent p21 expression. Additionally we induced DNA damage response in HCT116 wt and MEF cells by CPT-11 treatment and used HCT116 p21-/- cells a negative control. Unfortunately the antibody gives some unspecific signals at 20 kDa in HeLa cells blocked in G2 phase.Thus it's hard to distinguish between specific and unspecific signal in these cells. The antibody work well for the HCT116 cells. But does not work in murine cells. We used 15 ´g HeLa and MEF cell lysates, but only 7 ´g of HCT cell lysates.
Abcam response
Our Scientific Support team is currently investigating this negative Abreview, and we will resolve the issue in accordance with our Abpromise.

Mr. Christian Marx

Verified customer

Submitted Jan 15 2014

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