Anti-ROCK2 antibody [EPR7141(B)]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ROCK2 antibody [EPR7141(B)] (AB125025)

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling ROCK2 with purified ab125025 at 1/250. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab7291 anti-Tubulin (mouse mAb) primary and ab150120 (AlexaFluor^®594 goat anti-mouse) secondary both at 1/1000 dilution. Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody and ab150120 (anti-mouse) secondary antibody were used. For negative control 2, ab7291 (mouse primary antibody) was used followed by ab150077 (anti-rabbit secondary antibody).

• WB

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Western blot - Anti-ROCK2 antibody [EPR7141(B)] (AB125025)

All lanes:

Western blot - Anti-ROCK2 antibody [EPR7141(B)] (ab125025) at 1/10000 dilution

Lane 1:

Hela cell lysate at 10 µg

Lane 2:

L6 cell lysate at 10 µg

Lane 3:

A673 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 161 kDa

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• WB

Lab

Western blot - Anti-ROCK2 antibody [EPR7141(B)] (AB125025)

Lanes 1- 4 : Merged signal (red and green). Green - ab125025 observed at 175 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab125025 was shown to react with ROCK2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265679 (knockout cell lysate ab257643) was used. Wild-type HeLa and ROCK2 knockout 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. ab125025 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 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-ROCK2 antibody [EPR7141(B)] (ab125025) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ROCK2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ROCK2 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-rock2-knockout-hela-cell-line-ab265679'>ab265679</a>)

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 161 kDa

Observed band size: 175 kDa

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• WB

Lab

Western blot - Anti-ROCK2 antibody [EPR7141(B)] (AB125025)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : ROCK2 knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : A10 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab125025 observed at 165 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab125025 was shown to specifically react with ROCK2 when ROCK2 knockout samples were used. Wild-type and ROCK2 knockout samples were subjected to SDS-PAGE. ab125025 and ab8245 (loading control to GAPDH) were diluted 1/10 000 and 1/2000 respectively and incubated overnight at 4°C. 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 1/10,000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ROCK2 antibody [EPR7141(B)] (ab125025)

Predicted band size: 161 kDa

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• WB

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Western blot - Anti-ROCK2 antibody [EPR7141(B)] (AB125025)

Blocking and Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-ROCK2 antibody [EPR7141(B)] (ab125025) at 1/50000 dilution

Lane 1:

HeLa whole cell lysate at 10 µg

Lane 2:

HEK293 whole cell lysate at 10 µg

Lane 3:

Mouse brain tissue lysate at 10 µg

Lane 4:

Rat brain tissue lysate at 10 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: 161 kDa

Observed band size: 161 kDa

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• WB

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Western blot - Anti-ROCK2 antibody [EPR7141(B)] (AB125025)

Blocking and Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-ROCK2 antibody [EPR7141(B)] (ab125025) at 1/10000 dilution

All lanes:

RAW264.7 whole cell lysate at 10 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: 161 kDa,305 kDa

Observed band size: 161 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-ROCK2 antibody [EPR7141(B)] (AB125025)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• WB

CiteAb

Western blot - Anti-ROCK2 antibody [EPR7141(B)] (AB125025)

ROCK2 western blot using anti-ROCK2 antibody [EPR7141(B)] ab125025. Publication image and figure legend from Li, M. Z., Zhan, Y., et al., 2020, Front Physiol, PubMed 31920724.

ab125025 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab125025 please see the product overview.

Effect of XSEC or EE and their combination on the expressions of GAP-43, NogoA/NgR and RhoA/ROCK2 in MCAO rats. Quantitative analysis of the protein (A,C,E) and mRNA (B,D,F) levels of GAP-43, NogoA/NgR, and RhoA/ROCK2, respectively (one-way ANOVA followed by Bonferroni's post hoc test, n = 4 per group). #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle group.

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