Product nameHuman PAK2 knockout HeLa cell line
Parental Cell LineHeLa
Mutation descriptionKnockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 3 and 2 bp insertion in exon 3
Knockout validationSanger Sequencing, Western Blot (WB)
Tested applicationsSuitable for: WBmore details
Recommended control: Human wild-type HeLa cell line (ab255448). 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, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and 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
FunctionThe activated kinase acts on a variety of targets. Phosphorylates ribosomal protein S6, histone H4 and myelin basic protein. Full length PAK 2 stimulates cell survival and cell growth. The process is, at least in part, mediated by phosphorylation and inhibition of pro-apoptotic BAD. Caspase-activated PAK-2p34 is involved in cell death response, probably involving the JNK signaling pathway. Cleaved PAK-2p34 seems to have a higher activity than the CDC42-activated form.
Tissue specificityUbiquitously expressed. Higher levels seen in skeletal muscle, ovary, thymus and spleen.
Sequence similaritiesBelongs to the protein kinase superfamily. STE Ser/Thr protein kinase family. STE20 subfamily.
Contains 1 CRIB domain.
Contains 1 protein kinase domain.
modificationsFull length PAK 2 is autophosphorylated when activated by CDC42/p21. Following cleavage, both peptides, PAK-2p27 and PAK-2p34, become highly autophosphorylated, with PAK-2p27 being phosphorylated on serine and PAK-2p34 on threonine residues, respectively. Autophosphorylation of PAK-2p27 can occur in the absence of any effectors and is dependent on phosphorylation of Thr-402, because PAK-2p27 is acting as an exogenous substrate.
During apoptosis proteolytically cleaved by caspase-3 or caspase-3-like proteases to yield active PAK-2p34.
Ubiquitinated, leading to its proteasomal degradation.
PAK-2p34 is myristoylated.
Cellular localizationCytoplasm and Nucleus. Cytoplasm > perinuclear region. Membrane. Interaction with ARHGAP10 probably changes PAK-2p34 location to cytoplasmic perinuclear region. Myristoylation changes PAK-2p34 location to the membrane.
- Information by UniProt
KO cell lysates
Our Abpromise guarantee covers the use of ab264814 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: 58 kDa.|
All lanes : Anti-PAK2 antibody [EP796Y] (ab76293) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PAK2 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 58 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?
ab76293 was shown to react with PAK2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264814 (knockout cell lysate ab257573) was used. Wild-type HeLa and PAK2 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. ab76293 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 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.
Allel-1: 1 bp insertion in exon 3.
Allel-2: 2 bp insertion in exon 3.
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.
ab264814 has not yet been referenced specifically in any publications.