All lanes : Anti-ROCK2 antibody (ab71598) at 1 µg/mlLane 1 : Liver (Mouse) Tissue LysateLane 2 : SK N SH (Human neuroblastoma) Whole Cell LysateLane 3 : U2OS (Human osteosarcoma cell line) Whole Cell LysateLane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell LysateLane 5 : Liver (Rat) Tissue LysateLysates/proteins at 10 µg per lane.SecondaryGoat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilutionPerformed under reducing conditions.
![ROCK2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to ROCK2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab71598.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 171kDa; ROCK2](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_4/24247_ROCK2-Primary-antibodies-ab71598-24.jpg)
ROCK2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to ROCK2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab71598.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 171kDa; ROCK2
ICC/IF image of ab71598 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 (ab71598, 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 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.
All lanes : Anti-ROCK2 antibody (ab71598) at 1/1000 dilutionLane 1 : Human primary myometrial whole cell lysate with scrambled siRNALane 2 : Human primary myometrial whole cell lysate with scrambled siRNALane 3 : Human primary myometrial whole cell lysate with siRNA against ROCK2Lane 4 : Human primary myometrial whole cell lysate with siRNA against ROCK2Lysates/proteins at 20 µg per lane.SecondaryHRP conjugated Goat anti-rabbit IgG polyclonal at 1/4000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.