Nori Canine HIP-1 ELISA Kit Summary

Alternative names for HIP-1, HIP1, Huntingtin-interacting protein 1

Alternative names for canine: Dog

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:

Huntingtin-interacting protein 1 (HIP-1) is a protein that is encoded by the HIP1 gene.[1]

Hip-1 interacts with the huntingtin protein. HIP-1 contains a domain homologous to the death effector domains. It is believed that accumulation of high levels of the free form of HIP-1 in the cell is one of the mechanisms by which neuron cell death is caused in Huntington’s disease (via the caspase-3 route). The role of Hip-1 in caspase mediated cell death remains unclear. HIP1 was found to bind to Htt in an N-terminal dependent manner, and co-localizes with Htt in the CNS although the nature of this interaction with respect to muHtt was not identified. The CAG expansion seen with muHtt results in decreased binding affinity for HIP1, thus causing disruption of HIP1’s usual function, and also an increase in free HIP1.^[2] It is likely that this decreased affinity plays a role in mediating HD pathogenesis, due to loss of cytoskeletal integrity and induction of apoptosis. HIP1’s pro apoptotic effect may involve activation of caspase-8 and a novel HIP1 protein interactor HIPPI.^[3] HIP1’s non-pathological activity includes clathrin assembly via interaction with clathrin light chains.^[4] HIP1 is the human homologue of Sla2p, a membrane protein in the periphery.^[5] Actin binding by Hip-1 is altered depending on whether clathrin is also bound to Hip-1. HIP1 has been found to be overexpressed in some cancers including a subset of colorectal and prostate cancers.^[6] HIP1 can regulate transcription of hormone receptors via the androgen response element (ARE) and alter the rate of degradation of the AR.^[7]  HIP1 may regulate, or at least interact with proteins that also possess the ARE.

References

1. Wanker EE, et al. (1997). Human Molecular Genetics. 6(3): 487–95.
2. Hackam AS, et al. (2000). The Journal of Biological Chemistry. 275(52): 41299–308.
3. Gervais FG, et al. (2002). Nature Cell Biology. 4(2): 95–105.
4. Legendre-Guillemin V, et al. (2005). Journal of Biological Chemistry. 280(7): 6101–8.
5. Holzmann C, et al. (2001). Molecular Brain Research. 92(1–2): 85–97.
6. Rao DS, et al. (2002). The Journal of Clinical Investigation. 110(3): 351–60.
7. Mills IG, et al. (2005). The Journal of Cell Biology. 170(2): 191–200.