Product nameHuman ROCK2 knockout HeLa cell line
Parental Cell LineHeLa
Mutation descriptionKnockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 4 and 59 bp deletion in exon 4
Knockout validationSanger Sequencing, Western Blot (WB)
Tested applicationsSuitable for: WBmore details
Recommended control: Human wild-type HeLa cell line (ab255928). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: DMEM (High Glucose) + 10% FBS
Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80ºC. Storage at -80ºC may result in loss of viability.
1. Thaw the vial in 37ºC water bath approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 ml) to a 15 ml/50 ml conical sterile polypropylene centrifuge tube containing 8.4 ml pre-warmed culture medium, wash vial with an additional 0.8 ml culture medium (total volume 10 ml) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 ml represents minimum recommended dilution. 20 ml represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 ml pre-warmed culture medium and count using a haemocytometer (Click here to view haemocytometer protocol) or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. This should allow for confluency within 48 hours. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37ºC incubator with 5% CO2. Cultures should be monitored daily.
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended for confluency (80-90% confluence) within 48 hours.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
Number of cells1 x 106 cells/vial, 1 mL
STR AnalysisAmelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10
Antibiotic resistancePuromycin 1.00µg/ml
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% DMSO, 2% Cellulose, methyl ether
FunctionRegulates the assembly of the actin cytoskeleton. Promotes formation of stress fibers and of focal adhesion complexes. Plays a role in smooth muscle contraction.
Sequence similaritiesBelongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family.
Contains 1 AGC-kinase C-terminal domain.
Contains 1 PH domain.
Contains 1 phorbol-ester/DAG-type zinc finger.
Contains 1 protein kinase domain.
Contains 1 REM (Hr1) repeat.
modificationsPhosphorylated upon DNA damage, probably by ATM or ATR.
Cellular localizationCytoplasm. Cell membrane. Cytoplasmic, and associated with actin microfilaments and the plasma membrane.
- Information by UniProt
KO cell lysates
Our Abpromise guarantee covers the use of ab265679 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use at an assay dependent concentration. Predicted molecular weight: 161 kDa.|
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?
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.
Allele-1: 59 bp deletion in exon 4.
Allele-2: 1 bp deletion in exon 4.
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab265679 has not yet been referenced specifically in any publications.