All lanes : Anti-ETFA antibody [2B11AE8] (ab110316) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : ETFA knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution

Predicted band size: 35 kDa
Observed band size: 35 kDa

Lanes 1-2: Merged signal (red and green). Green - ab110316 observed at 35 kDa. Red - loading control ab52901 observed at  kDa.

 ab110316 Anti-ETFA antibody [2B11AE8]  was shown to specifically react with ETFA in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266513 (knockout cell lysate ab257943) was used. Wild-type and ETFA knockout samples were subjected to SDS-PAGE. ab110316 and Anti-beta Tubulin [EP1331Y] - Microtubule Marker (ab52901) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-ETFA antibody [2B11AE8] (ab110316) at 1 µg/ml

Lane 1 : Human heart tissue
Lane 2 : HepG2 whole cells
Lane 3 : Human liver mitochondria
Lane 4 : Bovine heart mitochondria
Lane 5 : Rat liver mitochondria
Lane 6 : Mouse liver mitochondria

Lysates/proteins at 10 µg per lane.

Predicted band size: 35 kDa

Immunocytochemistry image of stained HeLa cells. The cells were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15 minutes). The cells were incubated with the antibody (ab110316, 4 µg/mL) for 2 hours at room temperature or over night at 4°C. The secondary antibody was (red) Alexa Fluor® 594 goat anti-mouse IgG (H+L) at a 1/1000 dilution for 1 hour. 10% Goat serum was used as the blocking agent for all blocking steps. The target protein locates to the mitochondrial matrix

HL-60 cells were stained with 1 µg/mL ab110316 (blue) or an equal amount of an isotype control antibody (red) and analyzed by flow cytometry.

IHC image of ETFA staining in Human heart formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab110316, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.