All lanes : Anti-Calcineurin A antibody [EPR1670(2)] (ab109412) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PPP3CA knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 59 kDa
Observed band size: 59 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab109412 observed at 59 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109412 was shown to react with Calcineurin A in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265130 (knockout cell lysate ab257181) was used. Wild-type HeLa and PPP3CA 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. ab109412 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 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.

Immunofluorescent staining of HeLa cells using 1/100 ab109412.

Overlay histogram showing HeLa cells stained with ab109412 (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 (ab109412, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (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-Calcineurin A antibody [EPR1670(2)] (ab109412) at 1/3000 dilution

Lane 1 : Mouse brain cortex lysate
Lane 2 : Mouse lung lysate
Lane 3 : Rat brain cortex lysate
Lane 4 : Rat liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 59 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Calcineurin A knockout HAP1 cell lysate (20 µg)
Lane 3: A431 cell lysate (20 µg)
Lane 4: HeLa cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab109412 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109412 was shown to specifically react with Calcineurin A when Calcineurin A knockout samples were used. Wild-type and Calcineurin A knockout samples were subjected to SDS-PAGE. ab109412 and ab8245 (loading control to GAPDH) were both diluted 1/10000 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.

All lanes : Anti-Calcineurin A antibody [EPR1670(2)] (ab109412) at 1/10000 dilution

Lane 1 : fetal brain lysate
Lane 2 : SH-SY5Y lysate
Lane 3 : A431 lysate
Lane 4 : HeLa cells lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 59 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?