HMGB1, Mouse, mAb 5H6 Antibody

For flow cytometry, immunofluorescence and Western blot, dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50.

FC: For staining cells were permeabilized with buffer containing 0.5% saponin. The cells were fixed in 0.01% formaldehyde before staining. Hamster IgG isotype control is used as a negative control. IF: NIH-3T3 cells were fixed with 2% PFA (15 min) . After fixation, cells were washed with PBS and incubated for 5 min at 4°C with permeabilization buffer . The cells were blocked with 1% BSA/10% goat serum in PBS for 10 min and were incubated with HMGB1 mAb 5H6 (10 μg/ml) overnight at 4°C. The secondary antibody was biotinylated goat anti-hamster IgG at a 1:500 dilution for 1 hour. Finally, streptavidin was used at a 1:500 dilution for 30 min. W: A band is expected at ~30 kDa using reducing conditions. For non-reducing conditions a band is expected at both 27 kDa and 30 kDa.