Torsin A (Torsin family 1 member A, Dystonia 1 protein)

Torsin A, encoded by the DYT1 gene, is a member of the AAA family of adenosine triphosphatases (ATPases), associated with diverse cellular activities (Konakova et al., 2001).\n\nOzelius et al. (1997) constructed a cosmid contig spanning the region of chromosome 9 to which early-onset torsion dystonia (128100) had been mapped. Using exon trapping and identification of cDNAs encoded in this region, and mutational screening by SSCP and sequence analysis of cDNA and genomic DNA from affected individuals and controls, they identified the DYT1 gene. The deduced protein, termed 'torsinA' by them, comprises 332 amino acids and has a calculated molecular mass of 37.813kD. It contains an ATP-binding domain and a putative N-terminal leader sequence. DYT1 has high homology to 3 additional mammalian genes and a nematode gene and distal similarity to the family of heat-shock proteins and the Clp protease family. Northern blot analysis detected 2 ubiquitously expressed transcripts of 1.8kb and 2.2kb and an additional low-abundance transcript of 5kb in fetal brain, lung, and kidney, as well as in adult brain, heart, and pancreas.\n\nUsing an mRNA probe for DYT1 in normal human postmortem brains, Augood et al. (1998) found high expression in melanized neurons of the pars compacta of the substantia nigra, as well as in the cerebellum and the dentate gyrus and stratum pyramidal of CA3.\n\nUsing polyclonal antibodies directed against human torsin-A and torsin-B, Konakova et al. (2001) analyzed the expression of the proteins in normal human brain regions and found widespread neuronal distribution. Areas of intense labeling included the dentate nucleus and Purkinje cells of the cerebellum, all layers of the cerebral cortex, all subfields of the hippocampus, particularly CA3, the thalamus, the spinal cord, and the midbrain, and there was weak labeling in the substantia nigra. Staining was predominantly cytoplasmic, but also extended into the axons, dendrites, and neuropil. Glial cells showed no expression of the proteins. Torsin B showed a similar pattern of expression, but was found to have some polarization toward the cell edge, and was detected in the pigmented cells of the substantia nigra. Both proteins were present at high levels in regions not affected by disease (cortex, cerebellum, spinal cord). Konakova et al. (2001) noted that the staining pattern was granular and present in the neuronal processes, suggesting a role in regulating neurotransmitter release.\n\nApplications: \nSuitable for use in Western Blot, Immunoprecipitation and Immunocytochemistry. Other applications not tested.\n\nRecommended Dilution:\nImmunocytochemistry: 1:100 on 4% paraformaldehyde fixed (37 degrees C for 30 minutes) cells. Suggested permeabilization method is 0.1% NP40 for 20 minutes.\nImmunoprecipitation: The preferred tissue/cell lysis buffer is 1% NP40 and/or RIPA. Recommended antibody amount is 3-4ul of ascites. Final reaction volume of 1000ul. Final total protein concentration in reaction mix: 0.1-0.5mg/ml. Suggested incubation time is 16 hours. Sugggested capture is Protein A/G.\nWestern Blot: 1:200. Western blot: 1:200. The antibody reacts with the ~37 kDa torsinA protein. The suggested blocking buffer is TBS with 10% milk and 0.05% Tween-20. The suggested dilution buffer is TBS with 0.05% Tween-20. Recognizes Torsin A at ~3 7kD.\n\nOptimal dilutions to be determined by the researcher.\n\nStorage and Stability:\nMay be stored at 4 degrees C for short-term only. For long-term storage and to avoid repeated freezing and thawing, aliquot Freeze at -20 degrees C. Aliquots are stable for at least 12 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

T8067-37

Size

100ul

Applications

ICC, IP, WB

Hosts

Mouse

Reactivities

Hum, Mouse, Rat

Form

Supplied as a liquid. No preservative added

P Type

Mab

Purity

Ascites

Isotype

IgG1

References

Hewett, et al., Journal of Neuroscience Res (2003) 72:158-168.\nHewett, et al., Journal of Neurochem (2004) 89:1186-1194.\nKamm, C. et al., Journal of Biological Chemistry (2004) 279:19882-19892.\nBragg, D. et al., Neuroscience (2004) 125:651-661.

Additional Info

Recognizes human Torsin A