Anti-ROCK1 antibody [EPR638Y]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROCK1 antibody [EPR638Y] (AB134181)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue sections labeling ROCK1 with Purified ab134181 at 1 : 1200 (1.28 μg/ml). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ROCK1 antibody [EPR638Y] (AB134181)

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling ROCK1 with purified ab134181 at 1/150 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150081) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (blue).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROCK1 antibody [EPR638Y] (AB134181)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling ROCK1 with Purified ab134181 at 1 : 1200 (1.28 μg/ml). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• IP

Unknown

Immunoprecipitation - Anti-ROCK1 antibody [EPR638Y] (AB134181)

Purified ab134181 at 1/120 dilution (2μg) immunoprecipitating ROCK1 in HeLa whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab134181 + HeLa whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab134181 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 158 kDa

All lanes:

Immunoprecipitation - Anti-ROCK1 antibody [EPR638Y] (ab134181)

Predicted band size: 158 kDa

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• WB

Lab

Western blot - Anti-ROCK1 antibody [EPR638Y] (AB134181)

Lane 1 : Wild type HAP1 whole cell lysate (40 μg)
Lane 2 : ROCK1 knockout HAP1 whole cell lysate (40 μg)
Lane 3 : HEK293 whole cell lysate (20 μg)
Lane 4 : HeLa whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab134181 observed at 165 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab134181 was shown to specifically react with ROCK1 when ROCK1 knockout samples were used. Wild-type and ROCK1 knockout samples were subjected to SDS-PAGE. ab134181 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 500 dilution and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ROCK1 antibody [EPR638Y] (ab134181)

Predicted band size: 158 kDa

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• WB

Supplier Data

Western blot - Anti-ROCK1 antibody [EPR638Y] (AB134181)

All lanes:

Western blot - Anti-ROCK1 antibody [EPR638Y] (ab134181) at 1/1000 dilution

Lane 1:

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

Lane 2:

Mouse brain at 20 µg

Lane 3:

Rat brain at 20 µg

Secondary

All lanes:

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

Predicted band size: 158 kDa

Observed band size: 158 kDa

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• WB

Lab

Western blot - Anti-ROCK1 antibody [EPR638Y] (AB134181)

Lanes 1- 2 : Merged signal (red and green). Green - ab134181 observed at 160 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab134181 was shown to react with ROCK1 in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab264780 (CRISPR/Cas9 edited cell lysate ab257642) lane below 160kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and ROCK1 CRISPR/Cas9 edited 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. ab134181 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at a 1 in 1000 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-ROCK1 antibody [EPR638Y] (ab134181) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ROCK1 CRISPR/Cas9 edited HeLa cell lysate at 20 µg

Predicted band size: 158 kDa

Observed band size: 160 kDa

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