Application
Western blot
Review text: Human skin fibroblasts (HFF-1) were starved in serum-free medium overnight, and then were incubated 30 min in the presence of either 25 or 50 ng/ml active EGF protein, doses widely used in stem cell culture medium, to test whether EGFR downstream signaling pathway is able to be activated.
Sample: Human Cell lysate - whole cell (skin fibroblasts)
Loading amount: 20 µg
Specification: skin fibroblasts
Treatment: 25 and 50 ng/ml EGF for 30 min
Gel Running Conditions: Non-reduced Denaturing (10%)
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
Primary antibody (in addition to 'EGF protein (Active)')
Name: Non-Abcam antibody was used: phospho-p44/42 MAPK (Erk1/2)
Host species: Rabbit
Clonality: Polyclonal
Dilution: 1/1000
Secondary antibody
Name: Non-Abcam antibody was used: anti-rabbit 2nd Ab HRP
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution: 1/10000
Detection
Detection method: ECL
Exposure: 1 second(s)
Bands: Specific: 42/44 kDa
Additional data
Additional Notes: Incubation of human fibroblasts with active EGF protein for short time greatly activated EGFR signaling pathway, as the receptor underwent endocytosis and dramatic phosphorylation of p44/42 MAPK.
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Submitted Jun 25 2010