Anti-ROCK2 + ROCK1 antibody [EP786Y]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROCK2 + ROCK1 antibody [EP786Y] (AB45171)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid gland carcinoma tissue labelling ROCK2 + ROCK1 with unpurified ab45171.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ROCK2 + ROCK1 antibody [EP786Y] (AB45171)

Overlay histogram showing HeLa cells stained with unpurified ab45171 (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 (ab45171, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 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) (0.1μg/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-ROCK2 + ROCK1 antibody [EP786Y] (AB45171)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling ROCK2 + ROCK1 with purified ab45171 at 1/50. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150078, an Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control : primary antibody (1/50) and secondary antibody, ab150113, an Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/500).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROCK2 + ROCK1 antibody [EP786Y] (AB45171)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human adenocarcinoma of the colon tissue labelling ROCK2 + ROCK1 with purified ab45171 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IP

Unknown

Immunoprecipitation - Anti-ROCK2 + ROCK1 antibody [EP786Y] (AB45171)

ab45171 (purified) at 1/40 immunoprecipitating ROCK2 + ROCK1 in HeLa cell lysate. For western blotting, a peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/1000).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

Observed band size: 158 kDa

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• WB

Lab

Western blot - Anti-ROCK2 + ROCK1 antibody [EP786Y] (AB45171)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171) at 1/5000 dilution

Lane 1:

HeLa cell lysate - treated with Calyculin A at 20 µg

Lane 2:

HeLa cell lysate - treated with Camptothecin at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Lane 5:

Ramos cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Observed band size: 158 kDa

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• WB

Lab

Western blot - Anti-ROCK2 + ROCK1 antibody [EP786Y] (AB45171)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171) at 1/5000 dilution

Lane 1:

PC-12 cell lysate at 20 µg

Lane 2:

RAW264.7 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Observed band size: 158 kDa

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• WB

Supplier Data

Western blot - Anti-ROCK2 + ROCK1 antibody [EP786Y] (AB45171)

Loading control : Anti-6X His tag® antibody [EPR20547] (ab213204)

Blocking buffer and concentration : 5% NFDM/TBST

Exposure Times :

Lane 1 : 3 seconds

Lane 2 : 7 seconds

Lanes 1 - 2:

Western blot - Alexa Fluor® 488 Anti-ROCK2 + ROCK1 antibody [EP786Y] (<a href='/ko/products/primary-antibodies/alexa-fluor-488-rock2-rock1-antibody-ep786y-ab205432'>ab205432</a>) at 1/1000 dilution

Lanes 1 - 2:

Western blot - PE Anti-ROCK2 + ROCK1 antibody [EP786Y] (<a href='/ko/products/primary-antibodies/pe-rock2-rock1-antibody-ep786y-ab224972'>ab224972</a>) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171) at 1/1000 dilution

Lane 1:

Recombinant Human ROCK1 protein (aa 1114 to 1354) (30 kDa)

Lane 2:

Recombinant Human ROCK2 protein (aa 1132 to 1388) (30 kDa)

Secondary

All lanes:

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

Observed band size: 30 kDa

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