Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human TNF alpha.
(Peptide available as ab66578.)
Our Abpromise guarantee covers the use of ab66579 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 26 kDa (predicted molecular weight: 25 kDa).Can be blocked with Human TNF alpha peptide (ab66578).
Abcam recommends using 3% Milk as the blocking agent.
|ICC/IF||Use a concentration of 1 µg/ml.|
Mature THP-1 and Raw264.7 cells were treated with LPS (1 ug/ml) for 6 hours to stimulate production of TNF alpha. Brefeldin A (300 ng/ml) was added in the final 3 hours of incubation to inhibit golgi traffic.
ab66579 stained RAW246.7 cells. The cells were treated with PMA for 24 hours, LPS for 6 hours and Befreldin for the final 3 hours. The cells were then 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab66579 at 1µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.