Product nameHuman MAPK7 (ERK5) knockout HeLa cell line
Parental Cell LineHeLa
Mutation descriptionKnockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion 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, 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
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% DMSO, 2% Cellulose, methyl ether
FunctionPlays a role in various cellular processes such as proliferation, differentiation and cell survival. The upstream activator of MAPK7 is the MAPK kinase MAP2K5. Upon activation, it translocates to the nucleus and phosphorylates various downstream targets including MEF2C. EGF activates MAPK7 through a Ras-independent and MAP2K5-dependent pathway. May have a role in muscle cell differentiation. May be important for endothelial function and maintenance of blood vessel integrity. MAP2K5 and MAPK7 interact specifically with one another and not with MEK1/ERK1 or MEK2/ERK2 pathways.
Tissue specificityExpressed in many adult tissues. Abundant in heart, placenta, lung, kidney and skeletal muscle. Not detectable in liver.
Sequence similaritiesBelongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. MAP kinase subfamily.
Contains 1 protein kinase domain.
DomainThe second proline-rich region may interact with actin targeting the kinase to a specific location in the cell.
The TXY motif contains the threonine and tyrosine residues whose phosphorylation activates the MAP kinases.
modificationsDually phosphorylated on Thr-219 and Tyr-221, which activates the enzyme (By similarity). Autophosphorylated in vitro on threonine and tyrosine residues when the C-terminal part of the kinase, which could have a regulatory role, is absent.
Cellular localizationCytoplasm. Nucleus. Translocates to the nucleus upon activation.
- Information by UniProt
Our Abpromise guarantee covers the use of ab265508 in the following tested applications.
|WB||Use at an assay dependent concentration. Predicted molecular weight: 88 kDa.|
All lanes : Anti-ERK5 antibody [EP791Y] (ab40809) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MAPK7 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 88 kDa
Observed band size: 115 kDa why is the actual band size different from the predicted?
ab40809 was shown to react with ERK5 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265508 (knockout cell lysate ab258042) was used. Wild-type HeLa and MAPK7 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. ab40809 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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.
Homozygous: 1 bp insertion 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.
ab265508 has not yet been referenced specifically in any publications.