Application
Western blot
Sample
Human Cell lysate - whole cell (human fibroblast)
Loading amount
10000 cells
Specification
human fibroblast
Gel Running Conditions
Reduced Denaturing (4-20% acrylamide SDS-PAGE)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Other product details
Concentration
0.5 µg/ml
Incubation time
1 hour(s) and 0 minute(s) · Temperature: 25°C
Secondary antibody
Name
Non-Abcam antibody was used: anti-rabbit HRP
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Detection
Detection method
ECL
Exposure
10 minute(s) and 0 second(s)
Bands
Specific: 25? kDa Non-specific: 100, 95, 55, 54, 15, 10 kDa
Additional data
Additional Notes
was unsure if ~25 kD band was even DHFR. This Ab is very dirty and gives a lot of background under conditions where other antibodies give clean signal.
Abcam response
Thank you for your honest and thoughtful feedback. Abcam has not yet tested this on endogenous protein. I would like to recommend increasing the primary antibody incubation time to overnight, at 4C. This may help reduce non-specific bands and increase the specific signal. You may also want to try using BSA instead of milk to reduce background signal.
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 Apr 08 2009