Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Cell lysate - whole cell (Huh7 (liver))
Specification
Huh7 (liver)
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: 5% milk / PBS-T
Dilution
1/250
Secondary antibody
Name
Non-Abcam antibody was used: Sigma goat a-mouse HRP
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/5000
Detection
Detection method
West Femto
Bands
Specific: 138 kDa Non-specific: ~48 kDa
Exposure
5 minute(s) and 0 second(s)
Positive control
Huh7 whole cell lysate
Additional data
Additional Notes
Works fairly well detects the correct band at about 138 kDa, but also a smaller band around 47-48 kDa for which I have been unable to find an explanation (occurs in other cell types as well). Cells were lysed in RIPA buffer containing protease inhibitors (cocktail pill). All blocking/dilution was done in 5% milk/PBS-T. Blocking and secondary at RT for 1 hour, primary overnight at 4C.
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投稿 Apr 30 2014