All lanes : Anti-UQCRFS1/RISP antibody [5A5] (ab14746) at 0.5 µg/ml

Lane 1 : Hela Whole Cell Lysate - Hydroxyurea Treated
Lane 2 : Hela Whole Cell Lysate - Bleomycin Treated
Lane 3 : Hela Whole Cell Lysate - Staurosporine Treated

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Observed band size: 25 kDa why is the actual band size different from the predicted?

Human normal kidney (cortex). Staining is observed intracellular. Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus , at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for mouse for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

All lanes : Anti-UQCRFS1/RISP antibody [5A5] (ab14746) at 1 µg/ml

Lane 1 : Isolated mitochondria from human heart at 5 µg
Lane 2 : Isolated mitochondria from bovine heart at 1 µg
Lane 3 : Isolated mitochondria from rat heart at 10 µg
Lane 4 : Isolated mitochondria from mouse heart at 10 µg

Overlay histogram showing Raji cells stained with ab14746 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14746, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Raji cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.