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Key Product Details

Validated by

Biological Validation

Species Reactivity

Human, Mouse, Rat, Rabbit

Applications

Electron Microscopy, Immunoblotting, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Immunoprecipitation, Knockdown Validated, Knockout Validated, Peptide ELISA, SDS-Page, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free

Concentration

1.0 mg/ml

Product Summary for PINK1 Antibody - BSA Free

Immunogen

PINK1 antibody was developed using a synthetic peptide made to the human PINK1 protein sequence (between residues 175-250). [Swiss-Prot Q9BXM7]

Reactivity Notes

Use in Mouse reported in scientific literature (PMID:33775690). All species in which poly(GP) peptides are synthesized. Human reactivity reported in multiple pieces of scientific literature.

Localization

Localizes mostly in mitochondrion and the 2 proteolytic processed fragments (Topological domain 111-581) of 55 kDa and 48 kDa localize mainly in cytosol.

Specificity

Human PINK1 Antibody will be reactive to isoform 2.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

62.7 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for PINK1 Antibody - BSA Free

Western Blot Detection of PINK1 in Mitochondrial and Cytosolic Fractions

Western Blot Detection of PINK1 in Mitochondrial and Cytosolic Fractions

PINK1/Parkin-mediated mitophagy. Western Blot of mitochondrial and cytosolic fractions for PINK1, Parkin, and ubiquitin. E Quantitation of protein levels, normalized to VDAC and GAPDH, Ubiquitin: n = 6, PINK1: n = 5, Parkin: n = 8. R, post-reoxygenation. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33980811/) licensed under a CC-BY license.
Immunocytochemistry/Immunofluorescence Staining of PINK1 in Treated HeLa Cells

Immunocytochemistry/Immunofluorescence Staining of PINK1 in Treated HeLa Cells

Immunocytochemistry of PINK1 antibody (BC100-494 Lot G). HeLa cells were treated with valinomycin (1 uM for 24h) prior to being fixed in 10% buffered formalin for 10 min and permeabilized in 0.1% Triton X-100 in PBS for 10 min. Cells were incubated with BC100-494 at 20 ug/mL for 1h at room temperature, washed 3x in PBS and incubated with Alexa Fluor488 anti-rabbit secondary antibody. PINK1 (Green) was detected at the mitochondria. Tubulin (Red) was detected using an anti-tubulin antibody with an anti-mouse DyLight 550 secondary antibody. DNA (Blue) was counterstained with DAPI. Note: mitochondria staining might not be easily observed without treatment with valinomycin or CCCP.
Immunohistochemical Staining of PINK1 in Mouse Optic Nerve

Immunohistochemical Staining of PINK1 in Mouse Optic Nerve

Confocal microscopy analysis of the mitophagy initiation in the RPE cells by staining PINK1 and PARKIN. One-year-old WT and dKO mice focusing on the RPE cells in the vicinity of the optic nerve (a,e). PINK1 (b, red) and PARKIN (c, green) were double-stained and the merged image (d) was used to count the colocalized puncta from WT. Similarly, in dKO PINK1 (f, red) and PARKIN (g, green) were double-stained, and the merged image (h) was used to count the colocalized puncta. dKO = NFE2L2/PGC1a double knockout. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32183173/) licensed under a CC-BY license.

Applications for PINK1 Antibody - BSA Free

Application
Recommended Usage

Immunoblotting

reported in multiple pieces of scientific literature

Immunocytochemistry/ Immunofluorescence

1:50 - 1:200

Immunohistochemistry-Frozen

reported in scientific literature (PMID 31908016)

Immunohistochemistry-Paraffin

reported in scientific literature (PMID 25083992)

Immunoprecipitation

reported in scientific literature (PMID 22078885)

Knockout Validated

reported in scientific literature (PMID 31066324)

Peptide ELISA

1:100 - 1:2000

SDS-Page

reported in scientific literature (PMID 27846363)

Western Blot

1:500 - 1:2000
Application Notes
NOTE: It's recomended to use 1-5% w/v BSA in TBS with 0.1% Tween-20 for all incubations in WB. Specific bands are seen at 48, 55 and 63 kDa in Western Blot. In WB, this antibody has been used in valinomycin and CCCP treated HeLa whole cell lysate.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 14 reviews rated 4 using BC100-494 in the following applications:

Published Applications

Read 242 publications using BC100-494 in the following applications:

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: PINK1

Phosphatase and Tensin Homolog (PTEN) is a tumor suppressor which acts as an antagonist to phosphatidylinositol 3-kinase (PI3K) signaling. PTEN exerts enzymatic activity as a phosphatidylinositol-3,4,5-trisphosphate (PIP3) phosphatase, opposing PI3K activity by reducing availability of PIP3 to proliferating cells. Loss of PTEN function leads to elevated PIP3 and increased activation of PI3K/AKT signaling in many types of cancer.

PINK1 (PTEN induced putative kinase 1) protein contains a N-terminal mitochondrial targeting sequence, putative transmembrane helix, linker region, serine (Ser65)/threonine (Thr257) kinase domain and C-terminal segment. PINK1 is translated in the cytosol, then translocated to the outer mitochondrial membrane where it is rapidly cleaved and degraded as a part of normal mitochondrial function. In damaged (depolarized) mitochondria, PINK1 becomes stabilized and accumulates, resulting in the subsequent phosphorylation of numerous proteins on the mitochondrial surface.

When PINK1 is imported into the cell, mitochondrial processing peptidase, presenilin-associated rhomboid-like protease and AFG3L2 cleave PINK1 and tag it for the ubiquitin-proteasome pathway, keeping low PINK1 protein expression at basal conditions (1,2). Accumulation of PINK1 in mitochondria indicate damage. PINK1 maintains mitochondrial function/integrity, provides protection against mitochondrial dysfunction during cellular stress, and is involved in the clearance of damaged mitochondria via selective autophagy (mitophagy) (3). PINK1 has a theoretical molecular weight of 63 kDa and undergoes proteolytic processing to generate at least two cleaved forms (55 kDa and 42 kDa).

Ultimately PARK2 (E3 Ubiquitin Ligase Parkin) is recruited to the damaged mitochondria where it is activated by 1) PINK-mediated phosphorylation of PARK2 at serine 65, and 2) PARK2 interaction with phosphorylated ubiquitin (also phosphorylated by PINK1 on serine 65) (4,5). There is a strong interplay between Parkin and PINK1, where loss-of-function of human PINK1 results in mitochondrial pathology and can be rescued by Parkin (2,4,5). Mutations in either Parkin or PINK1 alter mitochondrial turnover, resulting in the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson's disease. Mutations in the PINK1 gene located within the PARK6 locus on chromosome 1p35-p36 have been identified in patients with early-onset Parkinson's disease (6).

References

1.Rasool, S., Soya, N., Truong, L., Croteau, N., Lukacs, G. L., & Trempe, J. F. (2018). PINK1 autophosphorylation is required for ubiquitin recognition. EMBO Rep, 19(4). doi:10.15252/embr.201744981

2.Shiba-Fukushima, K., Arano, T., Matsumoto, G., Inoshita, T., Yoshida, S., Ishihama, Y., . . . Imai, Y. (2014). Phosphorylation of mitochondrial polyubiquitin by PINK1 promotes Parkin mitochondrial tethering. PLoS Genet, 10(12), e1004861. doi:10.1371/journal.pgen.1004861

3.Vives-Bauza, C., Zhou, C., Huang, Y., Cui, M., de Vries, R. L., Kim, J., . . . Przedborski, S. (2010). PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc Natl Acad Sci U S A, 107(1), 378-383. doi:10.1073/pnas.0911187107

4.McWilliams, T. G., Barini, E., Pohjolan-Pirhonen, R., Brooks, S. P., Singh, F., Burel, S., . . . Muqit, M. M. K. (2018). Phosphorylation of Parkin at serine 65 is essential for its activation in vivo. Open Biol, 8(11). doi:10.1098/rsob.180108

5.Exner, N., Treske, B., Paquet, D., Holmstrom, K., Schiesling, C., Gispert, S., . . . Haass, C. (2007). Loss-of-function of human PINK1 results in mitochondrial pathology and can be rescued by parkin. J Neurosci, 27(45), 12413-12418. doi:10.1523/jneurosci.0719-07.2007

6.Valente, E. M., Bentivoglio, A. R., Dixon, P. H., Ferraris, A., Ialongo, T., Frontali, M., . . . Wood, N. W. (2001). Localization of a novel locus for autosomal recessive early-onset parkinsonism, PARK6, on human chromosome 1p35-p36. Am J Hum Genet, 68(4), 895-900. doi:10.1086/319522

Long Name

PTEN-induced Putative Kinase 1

Alternate Names

BRPK, PARK6, Anti-PINK1 Antibody, BRPK Antibody, Mitochondrial Antibody, PINK1 Antibody, PINK1 mouse, PINK1 polyclonal

Entrez Gene IDs

65018 (Human)

Gene Symbol

PINK1

UniProt

Product Documents for PINK1 Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for PINK1 Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Protocols

View specific protocols for PINK1 Antibody - BSA Free (BC100-494):

[[URL:https://www.novusbio.com/products/pink1-antibody_bc100-494]][[Caption:P… Antibody]]
Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain according to standard Ponceau S procedure (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot of the protein stain.
5. Block the membrane using 5% BSA for at least 1 hour.
6. Dilute anti-PINK1 primary antibody in 1-5% w/v BSA in TBS with 0.1% Tween-20 for 1 hour at room temperature.
7. Wash the membrane in wash buffer three times for 10 minutes each.
8. Incubate in diluted HRP-conjugated Rabbit secondary antibody in 1% BSA (as per manufacturers instructions) and incubate 1 hour at room temperature.
9. Wash the blot in wash buffer three times for 10 minutes each (this step can be repeated as required to reduce background).
10. Apply the detection reagent of choice in accordance with the manufacturers instructions.

FAQs for PINK1 Antibody - BSA Free

Showing  1 - 4 of 4 FAQs Showing All
  • Q: We recently purchased BC100-494 and the HEK293 cell extract to serve as a positive control. The most prominent band I got is approximately 120 kDa. However, after the cells were treated with DMSO, the most intense band became approximately 60 and 50 kDa (like the datasheet). Since there are some references that mention that PINK1 can form dimer or associate with other mitochondrial proteins, I am wondering if the reason of 120 kDa band is due to the strong binding of protein complex?

    A: It is most likely being picked up also at 120 kDa due to associations with other proteins. Our lab has not characterized this higher molecular weight band, but this seems like a likely explanation based on some publications I have found. One which you might find useful is The Mitochondrial Fusion-Promoting Factor Mitofusin Is a Substrate of the PINK1/Parkin Pathway. I recommend trying a negative control competition assay with BC100-494PEP. This blocking peptide can help you determine if this 120 kDa band is specific. You can find a protocol for this procedure that we recommend at the following link, peptide competition protocol.

  • Q: We would like to check toxicity of BC100-494. Could you inform us of the concentration of sodium azide?

    A: This product contains 0.05% NaN3 as the preservative.

  • Q: I ordered PINK1 antibody(BC100-494) at about six months ago. After it shipped to my country, it has been stored at -20 degrees Celsius in the agent company ever since. I recently called the agent to deliver it to me, and that's when I found this product has been wrongly stored according to the datasheet. So what I am worrying now is that does this influence the efficiency of this antibody? If it does, how much damage has been caused? What should I do to reduce the bad effects? Should I store this antibody by small packages?

    A: We recommend that BC100-494 is stored at 4C and that it should not be frozen. Unfortunately we have no testing data regarding the use of the antibody following -20C storage, and as such I cannot guarantee that it will perform optimally now that it has been frozen. I suggest that you try using the antibody as usual, but perhaps at a higher (more concentrated) dilution than recommended in case its potency has been lost during the freezing process. I would store it at 4C from now on, and not freeze it again once you have thawed it, since repeated freeze-that cycles can be detrimental to performance.

  • Q: Have you tried PINK1 antibody for immunoblotting in HEK293FT cells?

    A: We have not performed any testing of this antibody in HEK293FT cells specifically. However, PINK1 is a widely expressed protein, and I would expect it to be present in that particular cell line (it is expressed in HEK293); however, you will most likely want to run this along side controls to confirm.

Showing  1 - 4 of 4 FAQs Showing All
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