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: Rat liver tissue lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab66320 observed at 171 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab66320 was shown to recognize ROCK2 when ROCK2 knockout samples were used, along with additional cross-reactive bands. Wild-type and ROCK2 knockout samples were subjected to SDS-PAGE. ab66320 at a concentration of 2 µg/ml and ab18058 (loading control to Vinculin) at a dilution of 1/10000 were 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-ROCK2 antibody (ab66320) at 1 µg/ml

Lane 1 : Liver (Mouse) Tissue Lysate
Lane 2 : SK N BE (Human neuroblastoma) Whole Cell Lysate
Lane 3 : Human liver tissue lysate - total protein (ab29889)
Lane 4 : HeLa Whole Cell Lysate - Hydroxyurea Treated (48hr, 2uM)
Lane 5 : Y79 (Human retinoblastoma cell line) Whole Cell Lysate
Lane 6 : Liver (Rat) Tissue Lysate
Lane 7 : HeLa Whole Cell Lysate - Staurosporine Treated (24hr, 500nM)
Lane 8 : Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate
Lane 9 : Jurkat Whole Cell Lysate - Staurosporine Treated (24hr, 500nM)
Lane 10 : HeLa Whole Cell Lysate - Bleomycin Treated (20U/ml)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 161 kDa
Observed band size: 171 kDa why is the actual band size different from the predicted?
Additional bands at: 50 kDa, 95 kDa. We are unsure as to the identity of these extra bands.

ICC/IF image of ab66320 stained HeLa cells. The cells were 10% neutral buffered formalin fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab66320, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

IHC image of ab66320 staining ROCK2 in Human liver formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab66320, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.