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Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human ROCK2.
(Peptide available as ab71597.)
Our Abpromise guarantee covers the use of ab71598 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IP||Use a concentration of 5 µg/ml.|
|IHC-Fr||Use at an assay dependent concentration.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 171 kDa (predicted molecular weight: 161 kDa).Can be blocked with Human ROCK2 peptide (ab71597).|
|IHC-P||Use a concentration of 5 µg/ml.|
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 - ab71598 observed at 171 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab71598 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. ab71598 and ab8245 (loading control to GAPDH) were diluted 1 μg/mL 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 1/10000 dilution for 1 h at room temperature before imaging.
ab71598 staining ROCK2 in wild-type HAP1 cells (top panel) and ROCK2 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab71598 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
IHC image of ROCK2 staining in Human liver cancer formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab71598, 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.