All lanes : Anti-MTCO2 antibody [EPR3314] (ab79393) at 1/10000 dilution

Lane 1 : K562 cell lysate
Lane 2 : MCF7 cell lysate
Lane 3 : THP1 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 25 kDa
Observed band size: 21 kDa why is the actual band size different from the predicted?

ab79393 at 1/100 dilution staining Cytochrome C oxidase subunit II in Human Liver (A), Human Heart(B) and HeLa Cell Line(C) by Immunohistochemistry, Paraffin-embedded tissues. Detection method of A and B was HRP-conjugated anti-rabbit with DAB substrate used for staining.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

ICC/IF image of ab79393 stained HepG2 cells. The cells were 100% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab79393, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Purified ab79393 at 1/50 dilution (2µg) immunoprecipitating MTCO2 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab79393 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab79393 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 21 kDa

Overlay histogram showing HepG2 cells stained with ab79393 (red line). The cells were fixed with 4% paraformaldehyde (10 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 followed by the antibody (ab79393, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.