Anti-Calcineurin A antibody [EPR1670(2)]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Calcineurin A antibody [EPR1670(2)] (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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Calcineurin A antibody [EPR1670(2)] (AB109412)

Immunofluorescent staining of HeLa cells using 1/100 ab109412.

• WB

Lab

Western blot - Anti-Calcineurin A antibody [EPR1670(2)] (AB109412)

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:

Western blot - Anti-Calcineurin A antibody [EPR1670(2)] (ab109412)

Predicted band size: 59 kDa

Observed band size: 60 kDa

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• WB

Lab

Western blot - Anti-Calcineurin A antibody [EPR1670(2)] (AB109412)

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.

All lanes:

Western blot - Anti-Calcineurin A antibody [EPR1670(2)] (ab109412) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PPP3CA knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PPP3CA (Calcineurin A) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-ppp3ca-calcineurin-a-knockout-hela-cell-line-ab265130'>ab265130</a>)

Predicted band size: 59 kDa

Observed band size: 59 kDa

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• WB

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Western blot - Anti-Calcineurin A antibody [EPR1670(2)] (AB109412)

All lanes:

Western blot - Anti-Calcineurin A antibody [EPR1670(2)] (ab109412) at 1/10000 dilution

Lane 1:

fetal brain lysate at 10 µg

Lane 2:

SH-SY5Y lysate at 10 µg

Lane 3:

A431 lysate at 10 µg

Lane 4:

HeLa cells lysate at 10 µg

Predicted band size: 59 kDa

Observed band size: 58 kDa

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• WB

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Western blot - Anti-Calcineurin A antibody [EPR1670(2)] (AB109412)

All lanes:

Western blot - Anti-Calcineurin A antibody [EPR1670(2)] (ab109412) at 1/3000 dilution

Lane 1:

Mouse brain cortex lysate at 10 µg

Lane 2:

Mouse lung lysate at 10 µg

Lane 3:

Rat brain cortex lysate at 10 µg

Lane 4:

Rat liver lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 59 kDa

Observed band size: 58 kDa,59 kDa

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