Description

Nori Human PINK1 ELISA Kit Summary

Alternative names for PINK1: PTEN-induced kinase 1

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

PTEN-induced kinase 1 (PINK1) is a mitochondrial serine/threonine-protein kinase encoded by the PINK1 gene.^[1] PINK1 is synthesized as a 63 kDa protein which is often cleaved by PARL, between the 103-Alanine and the 104-Phenylalanine residues, into a 53 kDa fragment.^[2] PINK1 contains an N-terminal mitochondrial localization sequence, a putative transmembrane sequence, a Ser/Thr kinase domain, and a C-terminal regulatory sequence. The protein has been found to localize to the outer membrane of mitochondria, but can also be found throughout the cytosol. PARKL1 protects cells from stress-induced mitochondrial dysfunction. PINK1 activity causes the parkin protein to bind to depolarized mitochondria to induce autophagy of those mitochondria.^[3] PINK1 is processed by healthy mitochondria and released to trigger neuron differentiation.^[4] Mutations in this gene cause one form of autosomal recessive early-onset Parkinson’s disease. PINK1 is intimately involved with mitochondrial quality control by identifying damaged mitochondria and targeting specific mitochondria for degradation. Healthy mitochondria maintain a membrane potential that can be used to import PINK1 into the inner membrane where it is cleaved by PARL and cleared from the outer membrane. Severely damaged mitochondria lack sufficient membrane potential to import PINK1, which then accumulates on the outer membrane. PINK1 then recruits parkin to target the damaged mitochondria for degradation through autophagy. PINK1 has been implicated in mitochondrial motility. The accumulation of PINK1 and recruitment of parkin targets a mitochondria for degradation, and PINK1 may serve to enhance degradation rates by arresting mitochondrial motility. Over-expression of PINK1 produced similar effects to silencing Miro, a protein closely associated with mitochondrial migration.^[5]

References

1. Valente EM, et al. (2004). Ann Neurol. 56 (3): 336–41.
2. Deas E, et al. (2011). Hum. Mol. Genet. 20 (5): 867–869.
3. Lazarou M, et al. (2013). Journal of Cell Biology. 200(2): 163–172.
4. Dagda RK, et al. (2013). J Neurochem. 128 (6): 864–877.
5. Liu S, et al. (2012). PLoS Genetics. 8 (3): e102537. doi:1371/journal.pgen.1002537.

Product Citations