Application
Western blot
Sample
Human Cell lysate - whole cell (Human Aortic Endothelial Cell)
Loading amount
30 µg
Specification
Human Aortic Endothelial Cell
Treatment
200ng/ml VEGF
Gel Running Conditions
Reduced Denaturing (4-20% SDS-PAGE)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Other product details
Dilution
1/1000
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% Milk in TBST
Secondary antibody
Name
Non-Abcam antibody was used: anti-mouse HRP
Host species: Donkey
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Donkey
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/5000
Detection
Detection method
ECL+
Exposure
30 second(s)
Bands
Specific: 105-220 kDa
Positive control
+VEGF
Negative control
-VEGF
Additional data
Additional Notes
The antibody got a very dirty background for WB. Follow the suggestion and use milk instead of BSA but not help much. My sample is IP first and IB but still got a smear of beands at range 105kD to 220kD.
Abcam response
Thank you for your honest and thoughtful feedback. This antibody will detect all proteins in the cell with a phosphotyrosine. Therefore it is not surprising to see such large background on a lysate. In IP, it is usually not advisable to IP with the same antibody as the Western Blot. Immunoprecipitating with an antibody directed against your protein of interest, followed by a WB IP with ab10321, may improve the resuls you are seeing.
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.
Abcam user community
Verified customer
投稿 Aug 08 2008