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.

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).

All lanes : Anti-ROCK2 antibody [EPR7141(B)] (ab125025) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ROCK2 knockout HeLa cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 161 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

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

Lane 1 : HeLa whole cell lysate
Lane 2 : HEK293 whole cell lysate
Lane 3 : Mouse brain tissue lysate
Lane 4 : Rat brain tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 161 kDa
Observed band size: 161 kDa



Blocking and Diluting buffer 5% NFDM/TBST

All lanes : Anti-ROCK2 antibody [EPR7141(B)] (ab125025) at 1/10000 dilution (unpurified)

Lane 1 : Hela cell lysate
Lane 2 : L6 cell lysate
Lane 3 : A673 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 161 kDa

Anti-ROCK2 antibody [EPR7141(B)] (ab125025) at 1/10000 dilution + RAW264.7 whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 161 kDa
Observed band size: 161 kDa



Blocking and Diluting buffer 5% NFDM/TBST

Equilibrium disassociation constant (K_D)
Learn more about K_D

Click here to learn more about K_D