All lanes : Anti-Tropomyosin 3 antibody [3D5AH3AB4] (ab113692) 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 : TPM3 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 32 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?

Lanes 1-2: Merged signal (red and green). Green - ab113692 observed at 37 kDa. Red - loading control ab52901 observed at  kDa.

 ab113692 Anti-Tropomyosin 3 antibody [3D5AH3AB4] was shown to specifically react with Tropomyosin 3 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266422 (knockout cell lysate ab258245) was used. Wild-type and Tropomyosin 3 knockout samples were subjected to SDS-PAGE. ab113692 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.

Overlay histogram showing A431 cells stained with ab113692 (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 followed by the antibody (ab113692, 0.1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

All lanes : Anti-Tropomyosin 3 antibody [3D5AH3AB4] (ab113692) at 1 µg/ml

Lane 1 : HepG2 (human)
Lane 2 : HDFN (human)
Lane 3 : H9C2 (rat)
Lane 4 : H4IIE (rat)
Lane 5 : MEF (mouse)

Lysates/proteins at 20 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 32 kDa

ab113692 stained human fibroblast (HDFn) cells, rat cardiomyocytes (H9C2 cells) and mouse embryo fibroblast (MEF) cells. The cells were paraformaldehyde fixed (4%, 20 min) and Triton X-100 permeabilized (0.1%, 15 min). The cells were incubated with the antibody (3D5AH3AB4,5 µg/ml) for 2h at room temperature or over night at 4°C. The secondary antibody was (red) Alexa Fluor® 594 goat anti-mouse IgG2b (H+L) at a 1/1000 dilution for 1h. 10% Goat serum was used as the blocking agent for all blocking steps. The target protein locates to cytoskeleton.

IHC image of Tropomyosin 3 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 ab113692, 10µg/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.