Anti-ASK1 antibody [EP553Y] - BSA and Azide free

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ASK1 antibody [EP553Y] - BSA and Azide free (AB177030)

Unpurified ab45178 (1/10) staining human ASK1 in human lung carcinoma by immunohistochemistry using paraffin embedded tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45178).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ASK1 antibody [EP553Y] - BSA and Azide free (AB177030)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ASK1 with purified ab45178 at 1/100 dilution (10 μg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45178).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ASK1 antibody [EP553Y] - BSA and Azide free (AB177030)

Immunofluorescent staining of HeLa cells using unpurified ab45178 (1/10).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45178).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ASK1 antibody [EP553Y] - BSA and Azide free (AB177030)

Overlay histogram showing HeLa cells stained with unpurified ab45178 (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 (ab45178, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit DyLight® 488 (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. Acquisition of >5,000 events was performed.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45178).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ASK1 antibody [EP553Y] - BSA and Azide free (AB177030)

Immunocytochemistry/ Immunofluorescence analysis of RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling ASK1 with Purified ab45178 at 1 : 100 (9.9 μg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 μg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45178).

• WB

Lab

Western blot - Anti-ASK1 antibody [EP553Y] - BSA and Azide free (AB177030)

This data was developed using ab45178, the same antibody clone in a different buffer formulation.

Western blot : Rabbit Monoclonal[EP553Y] to ASK1 ab45178 staining at 1/2000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 150-170 kDa in Wild-type A549 cell lysates with no signal observed at this size in MAP3K5 knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-ASK1 antibody [EP553Y] (<a href='/en-us/products/primary-antibodies/ask1-antibody-ep553y-ab45178'>ab45178</a>) at 1/2000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human MAP3K5 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-map3k5-knockout-a549-cell-line-ab301060'>ab301060</a>) at 20 µg

Lane 3:

U-2 OS at 20 µg

Secondary

Lanes 1 - 3:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 3:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 155 kDa

Observed band size: 150-170 kDa,50 kDa

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