Product nameHuman PARK2 (Parkin) knockout SH-SY5Y cell line
See all Parkin lysates
Parental Cell LineSHSY-5Y
Mutation descriptionKnockout achieved by using CRISPR/Cas9, Homozygous: 77 bp deletion in exon 2
Knockout validationWestern Blot (WB)
Tested applicationsSuitable for: WBmore details
Recommended control: Human wild-type SHSY-5Y cell line (ab275475). 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: 1:1 mixture of EMEM and F-12K + 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 1-2x104 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.
- All seeding densities should be based on cell counts gained by established methods.
- These cells grow as a mixture of floating and adherent cells.
- Remove media containing floating cells and recover cells by centrifugation, detach cells using standard methods, combine with floating cells and transfer to a new culture flask.
- A guide seeding density of 1-2x104 cells/cm2 is recommended.
- Cells should be seeded at a density conducive to cell–cell communication to proliferate. If cells are seeded too sparsely, growth rate is reduced and cell death is high.
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
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
- Cell Biology
- Proteolysis / Ubiquitin
- Proteasome / Ubiquitin
- Ubiquitin E3 Enzymes
- RING Finger E3 Ligase
FunctionFunctions within a multiprotein E3 ubiquitin ligase complex, catalyzing the covalent attachment of ubiquitin moieties onto substrate proteins, such as BCL2, SYT11, CCNE1, GPR37, STUB1, a 22 kDa O-linked glycosylated isoform of SNCAIP, SEPT5, ZNF746 and AIMP2. Mediates monoubiquitination as well as 'Lys-48'-linked and 'Lys-63'-linked polyubiquitination of substrates depending on the context. Participates in the removal and/or detoxification of abnormally folded or damaged protein by mediating 'Lys-63'-linked polyubiquitination of misfolded proteins such as PARK7: 'Lys-63'-linked polyubiquitinated misfolded proteins are then recognized by HDAC6, leading to their recruitment to aggresomes, followed by degradation. Mediates 'Lys-63'-linked polyubiquitination of SNCAIP, possibly playing a role in Lewy-body formation. Mediates monoubiquitination of BCL2, thereby acting as a positive regulator of autophagy. Promotes the autophagic degradation of dysfunctional depolarized mitochondria. Mediates 'Lys-48'-linked polyubiquitination of ZNF746, followed by degradation of ZNF746 by the proteasome; possibly playing a role in role in regulation of neuron death. Limits the production of reactive oxygen species (ROS). Loss of this ubiquitin ligase activity appears to be the mechanism underlying pathogenesis of PARK2. May protect neurons against alpha synuclein toxicity, proteasomal dysfunction, GPR37 accumulation, and kainate-induced excitotoxicity. May play a role in controlling neurotransmitter trafficking at the presynaptic terminal and in calcium-dependent exocytosis. Regulates cyclin-E during neuronal apoptosis. May represent a tumor suppressor gene.
Tissue specificityHighly expressed in the brain including the substantia nigra. Expressed in heart, testis and skeletal muscle. Expression is down-regulated or absent in tumor biopsies, and absent in the brain of PARK2 patients. Overexpression protects dopamine neurons from kainate-mediated apoptosis. Found in serum (at protein level).
PathwayProtein modification; protein ubiquitination.
Involvement in diseaseDefects in PARK2 are a cause of Parkinson disease (PARK) [MIM:168600]. A complex neurodegenerative disorder characterized by bradykinesia, resting tremor, muscular rigidity and postural instability. Additional features are characteristic postural abnormalities, dysautonomia, dystonic cramps, and dementia. The pathology of Parkinson disease involves the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies (intraneuronal accumulations of aggregated proteins), in surviving neurons in various areas of the brain. The disease is progressive and usually manifests after the age of 50 years, although early-onset cases (before 50 years) are known. The majority of the cases are sporadic suggesting a multifactorial etiology based on environmental and genetic factors. However, some patients present with a positive family history for the disease. Familial forms of the disease usually begin at earlier ages and are associated with atypical clinical features.
Defects in PARK2 are the cause of Parkinson disease type 2 (PARK2) [MIM:600116]; also known as early-onset parkinsonism with diurnal fluctuation (EPDF) or autosomal recessive juvenile Parkinson disease (PDJ). A neurodegenerative disorder characterized by bradykinesia, rigidity, postural instability, tremor, and onset usually befor 40. It differs from classic Parkinson disease by early DOPA-induced dyskinesia, diurnal fluctuation of the symptoms, sleep benefit, dystonia and hyper-reflexia. Dementia is absent. Pathologically, patients show loss of dopaminergic neurons in the substantia nigra, similar to that seen in Parkinson disease; however, Lewy bodies (intraneuronal accumulations of aggregated proteins) are absent.
Note=Defects in PARK2 may be involved in the development and/or progression of ovarian cancer.
Sequence similaritiesBelongs to the RBR family. Parkin subfamily.
Contains 1 IBR-type zinc finger.
Contains 2 RING-type zinc fingers.
Contains 1 ubiquitin-like domain.
DomainThe ubiquitin-like domain binds the PSMD4 subunit of 26S proteasomes.
modificationsAuto-ubiquitinates in an E2-dependent manner leading to its own degradation. Also polyubiquitinated by RNF41 for proteasomal degradation.
S-nitrosylated. The inhibition of PARK2 ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in PD by impairing the ubiquitination of PARK2 substrates.
Cellular localizationCytoplasm > cytosol. Nucleus. Endoplasmic reticulum. Mitochondrion. Mainly localizes in the cytosol. Co-localizes with SYT11 in neutrites. Co-localizes with SNCAIP in brainstem Lewy bodies. Relocates to dysfunctional mitochondria that have lost the mitochondial membrane potential; recruitement to mitochondria is PINK1-dependent.
- Information by UniProt
KO cell lysates
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab280042 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration. Predicted molecular weight: 52 kDa.
Use at an assay dependent concentration. Predicted molecular weight: 52 kDa.
All lanes : Anti-Parkin antibody [PRK8] (ab77924) at 5 µg/ml
Lane 1 : Wild-type SH-SY5Y cell lysate
Lane 2 : PRKN knockout SH-SY5Y cell lysate
Lane 3 : Human Brain tissue lysate
Lane 4 : HUVEC cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 52 kDa
Observed band size: 49 kDa why is the actual band size different from the predicted?
ab77924 was shown to react with Parkin in wild-type SH-SY5Y cells in Western blot with loss of signal observed in PRKN knockout cell line ab280042 (PRKN knockout cell lysate ab280101). Wild-type SH-SY5Y and PRKN knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab77924 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4 °C at 5 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
Human PRKN KO in SH-SY5Y Cells with 77 bp Deletion in Exon 2
ab280042 has not yet been referenced specifically in any publications.