All lanes : Anti-WSTF antibody [EP1704Y] (ab51256) at 1/15000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : WSTF knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 171 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab51256 observed at 175 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab51256 was shown to react with WSTF in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264907 (knockout cell lysate ab257370) was used. Wild-type HeLa and WSTF knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab51256 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 15000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast cancer tissue labelling WSTF with ab51256 at a dilution of 1/500. Heat mediated antigen retrieval was performed using citrate buffer, pH 6.0. A ready to use HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody. Counter stained with hematoxylin.

All lanes : Anti-WSTF antibody [EP1704Y] (ab51256) at 1/15000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : WSTF knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : PC12 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 171 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab51256 observed at 175 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab51265 was shown to recognize WSTF in wilt-type cells along with additional cross-reactive bands as signal was lost in WSTF knockout samples. Wild-type and WSTF knockout samples were subjected to SDS-PAGE. ab51256 and ab18058 (loading control to vinculin) were diluted 1/15 000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling WSTF with ab51256 at a dilution of 1/1000. Cells were fized with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG was used as the secondary antibody at a dilution of 1/1000.

Overlay histogram showing HeLa cells stained with ab51256 (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 (ab51256, 1/100 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. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cardiac musle tissue labelling WSTF with ab51256 at a dilution of 1/500. Heat mediated antigen retrieval was performed using citrate buffer, pH 6.0. A ready to use HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody. Counter stained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cardiac muscle tissue labelling WSTF with ab51256 at a dilution of 1/500. Heat mediated antigen retrieval was performed using citrate buffer, pH 6.0. A ready to use HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody. Counter stained with hematoxylin.

Anti-WSTF antibody [EP1704Y] (ab51256) at 1/20000 dilution + HeLa cell lysate at 10 µg

Secondary
Goat anti-Rabbit HRP labeled. at 1/2000 dilution

Predicted band size: 171 kDa
Observed band size: 185 kDa why is the actual band size different from the predicted?