Human MAPK1 (ERK2) knockout HeLa cell line (ab265052)
Overview
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Product name
Human MAPK1 (ERK2) knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 2 and Insertion of the selection cassette in exon 2 -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: ICC/IF, WBmore details -
Biosafety level
2 -
General notes
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 -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for 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 or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.Subculture guidelines:
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- 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.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin 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 -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
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Function
Involved in both the initiation and regulation of meiosis, mitosis, and postmitotic functions in differentiated cells by phosphorylating a number of transcription factors such as ELK1. Phosphorylates EIF4EBP1; required for initiation of translation. Phosphorylates microtubule-associated protein 2 (MAP2). Phosphorylates SPZ1 (By similarity). Phosphorylates heat shock factor protein 4 (HSF4) and ARHGEF2.
Acts as a transcriptional repressor. Binds to a [GC]AAA[GC] consensus sequence. Repress the expression of interferon gamma-induced genes. Seems to bind to the promoter of CCL5, DMP1, IFIH1, IFITM1, IRF7, IRF9, LAMP3, OAS1, OAS2, OAS3 and STAT1. Transcriptional activity is independent of kinase activity. -
Sequence similarities
Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. MAP kinase subfamily.
Contains 1 protein kinase domain. -
Domain
The TXY motif contains the threonine and tyrosine residues whose phosphorylation activates the MAP kinases. -
Post-translational
modificationsDually phosphorylated on Thr-185 and Tyr-187, which activates the enzyme. Dephosphorylated by PTPRJ at Tyr-187. -
Cellular localization
Nucleus. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab265052 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| ICC/IF |
Use at an assay dependent concentration.
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| WB |
Use at an assay dependent concentration. Predicted molecular weight: 41 kDa.
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| Notes |
|---|
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ICC/IF
Use at an assay dependent concentration. |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 41 kDa. |
Images
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Immunocytochemistry/ Immunofluorescence - Human MAPK1 (ERK2) knockout HeLa cell line (ab265052)
ab32081 staining ERK2 in wild-type HeLa cells (top panel) and ERK2 knockout HeLa cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween 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 overnight at 4°C with ab32081 at 0.2µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
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Western blot - Human MAPK1 knockout HeLa cell line (ab265052)All lanes : Anti-ERK1 + ERK2 antibody [EPR17526] (ab184699) at 1/10000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MAPK1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 41 kDa
Observed band size: 44 kDa why is the actual band size different from the predicted?Lanes 1-2: Merged signal (red and green). Green - ab184699 observed at 44 kDa. Red - loading control ab8245 observed at 37 kDa.
ab184699 Anti-ERK1 + ERK2 antibody [EPR17526] was shown to specifically react with ERK2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265052 (knockout cell lysate ab257525) was used. Wild-type and ERK2 knockout samples were subjected to SDS-PAGE. ab184699 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 Dilution and 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.
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Western blot - Human MAPK1 knockout HeLa cell line (ab265052)All lanes : Anti-ERK2 antibody [E460] (ab32081) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MAPK1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 41 kDa
Observed band size: 41 kDaLanes 1-2: Merged signal (red and green). Green - ab32081 observed at 41 kDa. Red - loading control ab8245 observed at 37 kDa.
ab32081 Anti-ERK2 antibody [E460] was shown to specifically react with ERK2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265052 (knockout cell lysate ab257525) was used. Wild-type and ERK2 knockout samples were subjected to SDS-PAGE. ab32081 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 Dilution and 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.
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Sanger Sequencing - Human MAPK1 knockout HeLa cell line (ab265052)Allele-1: 1 bp deletion in exon 2.
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Sanger Sequencing - Human MAPK1 knockout HeLa cell line (ab265052)Allele-2: Insertion of the selection cassette in exon 2.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab265052 has not yet been referenced specifically in any publications.