Knockout Tested Rabbit Recombinant Monoclonal PINK1 antibody. Suitable for IP, WB, ICC/IF and reacts with Human samples. Cited in 20 publications.
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
IP | WB | ICC/IF | |
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Human | Tested | Tested | Tested |
Species | Dilution info | Notes |
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Species Human | Dilution info 1/30 | Notes - |
Species | Dilution info | Notes |
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Species Human | Dilution info 1/1000 | Notes - |
Species | Dilution info | Notes |
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Species Human | Dilution info 1/500 | Notes - |
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Serine/threonine-protein kinase which acts as a sensor of mitochondrial damage and protects against mitochondrial dysfunction during cellular stress. It phosphorylates mitochondrial proteins to coordinate mitochondrial quality control mechanisms that remove and replace dysfunctional mitochondrial components (PubMed:14607334, PubMed:15087508, PubMed:18443288, PubMed:18957282, PubMed:19229105, PubMed:19966284, PubMed:20404107, PubMed:20547144, PubMed:20798600, PubMed:22396657, PubMed:23620051, PubMed:23754282, PubMed:23933751, PubMed:24660806, PubMed:24751536, PubMed:24784582, PubMed:24896179, PubMed:24898855, PubMed:25527291, PubMed:32484300). Depending on the severity of mitochondrial damage, activity ranges from preventing apoptosis and stimulating mitochondrial biogenesis to eliminating severely damaged mitochondria via PINK1-PRKN-dependent mitophagy (PubMed:14607334, PubMed:15087508, PubMed:18443288, PubMed:19966284, PubMed:20404107, PubMed:20798600, PubMed:22396657, PubMed:23620051, PubMed:23933751, PubMed:24898855, PubMed:32047033, PubMed:32484300). When cellular stress results in irreversible mitochondrial damage, PINK1 accumulates at the outer mitochondrial membrane (OMM) where it phosphorylates pre-existing polyubiquitin chains at 'Ser-65', recruits PRKN from the cytosol to the OMM and activates PRKN by phosphorylation at 'Ser-65'; activated PRKN then ubiquinates VDAC1 and other OMM proteins to initiate mitophagy (PubMed:14607334, PubMed:15087508, PubMed:19966284, PubMed:20404107, PubMed:20798600, PubMed:23754282, PubMed:23933751, PubMed:24660806, PubMed:24751536, PubMed:24784582, PubMed:25474007, PubMed:25527291, PubMed:32047033). The PINK1-PRKN pathway also promotes fission of damaged mitochondria through phosphorylation and PRKN-dependent degradation of mitochondrial proteins involved in fission such as MFN2 (PubMed:18443288, PubMed:23620051, PubMed:24898855). This prevents the refusion of unhealthy mitochondria with the mitochondrial network or initiates mitochondrial fragmentation facilitating their later engulfment by autophagosomes (PubMed:18443288, PubMed:23620051). Also promotes mitochondrial fission independently of PRKN and ATG7-mediated mitophagy, via the phosphorylation and activation of DNM1L (PubMed:18443288, PubMed:32484300). Regulates motility of damaged mitochondria by promoting the ubiquitination and subsequent degradation of MIRO1 and MIRO2; in motor neurons, this likely inhibits mitochondrial intracellular anterograde transport along the axons which probably increases the chance of the mitochondria undergoing mitophagy in the soma (PubMed:22396657). Required for ubiquinone reduction by mitochondrial complex I by mediating phosphorylation of complex I subunit NDUFA10 (By similarity). Phosphorylates LETM1, positively regulating its mitochondrial calcium transport activity (PubMed:29123128).
BRPK, PTEN-induced putative kinase protein 1, PINK1
Knockout Tested Rabbit Recombinant Monoclonal PINK1 antibody. Suitable for IP, WB, ICC/IF and reacts with Human samples. Cited in 20 publications.
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
The PTEN-induced kinase 1 (PINK1) is a serine/threonine-protein kinase with a molecular weight of approximately 63 kDa. It plays a significant role in mitochondrial quality control through its kinase activity. PINK1 gets expressed in various tissues with a high presence in the brain. The protein localizes to the outer membrane of damaged mitochondria to initiate mitophagy an important cellular process for clearing dysfunctional mitochondria.
The PINK1 protein detects mitochondrial damage and recruits Parkin an E3 ubiquitin ligase to the damaged mitochondria. This interaction leads to the ubiquitylation of mitochondrial substrates and triggers their degradation. PINK1 Parkin and other proteins form a complex that facilitates the removal of damaged mitochondria through autophagy. This mechanism ensures cellular health and energy balance by maintaining a pool of functional mitochondria.
PINK1 integrates into the mitochondrial quality control and mitophagy pathways. It has a fundamental role in the PINK1-Parkin pathway which is critical for maintaining mitochondrial integrity. In this pathway PINK1 phosphorylates both ubiquitin and Parkin enhancing Parkin’s E3 ligase activity. Another pathway that PINK1 participates in is the PTEN-induced kinase pathway where it regulates mitochondrial dynamics and homeostasis via cross-talk with other mitochondrial proteins such as DJ-1 and LRRK2.
PINK1 mutations are strongly associated with familial Parkinson’s disease attributing to its role in preserving neuronal function through mitochondrial regulation. Deficient PINK1 function leads to accumulation of damaged mitochondria contributing to neuronal cell death. Additionally PINK1 dysfunction is implicated in Alzheimer’s disease as impaired mitochondrial clearance is a contributing factor. Here the PINK1 interaction with other proteins like Parkin and DJ-1 highlights its importance in neurodegenerative disorders.
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Dilution/blocking buffer: 5% NFDM/TBST
All lanes: Western blot - Anti-PINK1 antibody [EPR20730] (ab216144) at 1/1000 dilution
All lanes: Human PINK1 recombinant protein (aa156-507), 10 ng
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/50000 dilution
Developed using the ECL technique.
Predicted band size: 63 kDa
Observed band size: 38 kDa
Exposure time: 1s
Immunofluorescent analysis of 4 % paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma)(+/- treatment with 10μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229) for 24 hours) cells labeling PINK1 with ab216144 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cells treated with 10μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229) for 24 hours. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889) at 1/200 dilution (red).
The negative controls are as follows:
-ve control: PBS, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution.
PINK1 can be induced by CCCP treatment (PMID: 24184327).
All lanes: Western blot - Anti-PINK1 antibody [EPR20730] (ab216144) at 1/1000 dilution
Lane 1: HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: HeLa cells (treated with 10uM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229) for 24 hours) whole cell lysate at 20 µg
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Developed using the ECL technique.
Predicted band size: 63 kDa
Observed band size: 62 kDa
Exposure time: 5s
PINK1 was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) (treated with 10uM carbonyl cyanide 3-chlorophenylhydrazone (CCCP. CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229) for 24 hours) whole cell lysate with ab216144 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab216144 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/1,000 dilution.
Lane 1: HeLa (CCCP-treated, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229) lysate 10 μg (Input).
Lane 2: ab216144 IP in HeLa (CCCP-treated, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229) lysate.
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab216144 in HeLa (CCCP-treated, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229) whole cell lysate.
Blocking and dilution buffer: 5% NFDM/TBST.
All lanes: Immunoprecipitation - Anti-PINK1 antibody [EPR20730] (ab216144)
Developed using the ECL technique.
Predicted band size: 63 kDa
Observed band size: 62 kDa
Exposure time: 5s
Anti-PINK1 antibody [EPR20730] (ab216144) staining at 1/200 dilution shown in black; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. ab216144 was shown to bind specifically to PINK1. A band was observed at 60 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in PINK1 knockout cell line Human PINK1 knockout HEK-293T cell line ab266393 (knockout cell lysate ab257030). Membranes were blocked in 5 % milk in TBS-0.1 % Tween 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T and incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with Optiblot (ECL reagent Anti-PKC delta (phospho S299) antibody [EPNCI119] ab133456) and imaged with 20 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-PINK1 antibody [EPR20730] (ab216144) at 1/200 dilution
Lane 1: Wild-type HEK-293 Vehicle Control CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (0 µM, 24h) cell lysate at 20 µg
Lane 2: Wild-type HEK-293 Treated CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (10 µM, 24 h) cell lysate at 20 µg
Lanes 3 - 4: Western blot - Human PINK1 knockout HEK-293T cell line (Human PINK1 knockout HEK-293T cell line ab266393)
Lane 3: PINK1 knockout HEK-293 Vehicle Control CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (0 µM, 24 h) cell lysate at 20 µg
Lane 4: PINK1 knockout HEK-293 Treated CCCP, CCCP, Mitochondrial oxidative phosphorylation uncoupler ab141229 (10 µM, 24 h) cell lysate at 20 µg
All lanes: HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 63 kDa
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