Anti-MST3 antibody [EP1468Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MST3 antibody [EP1468Y] (AB51137)

Immunofluorescent staining of HeLa cells using ab51137 (1 : 100).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MST3 antibody [EP1468Y] (AB51137)

Overlay histogram showing HeLa cells stained with ab51137 (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 (ab51137 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 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). Acquisition of >5000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MST3 antibody [EP1468Y] (AB51137)

ab51137 (1 : 100) staining human MST3 in human brain tissue by immunohistochemistry using paraffin embedded tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

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Western blot - Anti-MST3 antibody [EP1468Y] (AB51137)

All lanes:

Western blot - Anti-MST3 antibody [EP1468Y] (ab51137) at 1/5000 dilution

All lanes:

HeLa cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP labeled at 1/2000 dilution

Predicted band size: 49 kDa

Observed band size: 52 kDa

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• WB

Lab

Western blot - Anti-MST3 antibody [EP1468Y] (AB51137)

Lanes 1 - 4 : Merged signal (red and green). Green - ab51137 observed at 52 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab51137 was shown to react with MST3 in wild-type A431 cells in Western blot with loss of signal observed in STK24 knockout cell line ab269479 (knockout cell lysate ab269643). Wild-type A431 and STK24 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab51137 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-MST3 antibody [EP1468Y] (ab51137) at 1/1000 dilution

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

STK24 knockout A431 cell lysate at 20 µg

Lane 2:

Western blot - Human STK24 knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-stk24-knockout-a-431-cell-line-ab269479'>ab269479</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Raji cell lysate at 20 µg

Predicted band size: 49 kDa

Observed band size: 52 kDa

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