Application
Western blot
Sample
Human Cell lysate - whole cell (HCT116 and fibroblast cell lines)
Loading amount
100000 cells
Specification
HCT116 and fibroblast cell lines
Treatment
2 mM HU 16 hours
Gel Running Conditions
Reduced Denaturing (7-15% gradiant)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Other product details
Dilution
1/200
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 3% BSA in TBST
Secondary antibody
Name
Non-Abcam antibody was used: HRP conjugated donkey anti-rabbit
Host species: Donkey
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Donkey
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Detection
Detection method
PE Western lightening ECL+
Exposure
15 minute(s) and 0 second(s)
Positive control
Anti-gamma-H2AX and anti-HSC70 for loading and phosphorylation status
BLM+ normal cells vs. BLM-negative Bloom's syndrome cells
BLM+ normal cells vs. BLM-negative Bloom's syndrome cells
Negative control
No HU treatment is negative control
Additional data
Additional Notes
1. HepG2 cells should be a good control for WB.
We did not use HepG2 cells however, there is plenty of detectable BLM in the HCT116 and fibroblast cells lines we tested. We have published detailed analyses of the fibroblast cell lines -- for example, see PMIDs 15829507 and 11406610 -- and we have unpublished western analysis of the HCT116 cell lines.
We chose these particular cell lines because we had comparable or isogenic BLM-negative cell lines that would allow us to distinguish cognate from non-specific bands.
2. Have you tried staining with other blocking agents such as BSA?
We did not try another blocking agent because the problem we experienced was lack of signal rather than high background.
We did not use HepG2 cells however, there is plenty of detectable BLM in the HCT116 and fibroblast cells lines we tested. We have published detailed analyses of the fibroblast cell lines -- for example, see PMIDs 15829507 and 11406610 -- and we have unpublished western analysis of the HCT116 cell lines.
We chose these particular cell lines because we had comparable or isogenic BLM-negative cell lines that would allow us to distinguish cognate from non-specific bands.
2. Have you tried staining with other blocking agents such as BSA?
We did not try another blocking agent because the problem we experienced was lack of signal rather than high background.
Abcam response
We appreciate customer's collaboration in testing the antibody in WB.
We welcome any other feedback regarding WB for ab62206
We welcome any other feedback regarding WB for ab62206
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.
PROF. Nathan Ellis
Verified customer
投稿 May 19 2011