Anti-Cystathionase/CTH antibody [EPR15468] - N-terminal

• IP

Supplier Data

Immunoprecipitation - Anti-Cystathionase/CTH antibody [EPR15468] - N-terminal (AB189916)

Immunoprecipitation of Cystathionase/CTH from HeLa cell lysate using ab189916 at 1/50 or 1X PBS (negative control) (lane 1). Immunostained with ab189916 at 1/1500 and HRP-conjugated anti-rabbit IgG preferentially detecting the non-reduced form of rabbit IgG.

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Immunoprecipitation - Anti-Cystathionase/CTH antibody [EPR15468] - N-terminal (ab189916)

Predicted band size: 45 kDa

Observed band size: 45 kDa

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• WB

Lab

Western blot - Anti-Cystathionase/CTH antibody [EPR15468] - N-terminal (AB189916)

Blocking and diluting buffer : 5% NFDM/TBST.

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Western blot - Anti-Cystathionase/CTH antibody [EPR15468] - N-terminal (ab189916) at 0.01 µg/mL

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HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

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Goat Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/20000 dilution

Predicted band size: 45 kDa

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Exposure time: 1min

• WB

Lab

Western blot - Anti-Cystathionase/CTH antibody [EPR15468] - N-terminal (AB189916)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : Cystathionase/CTH knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : K562 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab189916 observed at 45 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab189916 was shown to recognize Cystathionase/CTH in wild type cells as signal was lost at the expected MW in Cystathionase/CTH knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Cystathionase/CTH knockout samples were subjected to SDS-PAGE. ab189916 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Cystathionase/CTH antibody [EPR15468] - N-terminal (ab189916)

Predicted band size: 45 kDa

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• WB

Lab

Western blot - Anti-Cystathionase/CTH antibody [EPR15468] - N-terminal (AB189916)

Western blot : Anti-CTH antibody [EPR15468] (ab189916) staining at 1/10000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab189916 was shown to bind specifically to CTH. A band was observed at 42 kDa in wild-type A549 cell lysates with no signal observed at this size in CTH knockout cell line. To generate this image, wild-type and CTH knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Cystathionase/CTH antibody [EPR15468] - N-terminal (ab189916) at 1/10000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

CTH knockout A549 cell lysate at 20 µg

Lane 3:

K562 cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 42 kDa

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