Human SPG3A Baculovirus-Insect cells Overexpression Lysate

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Human SPG3A Baculovirus-Insect cells Overexpression Lysate: Product Information

Product Description
This Human SPG3A overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of SPG3A protein (Cat: 10523-H09B) from the overexpression lysate was verified.
Expression Host
Baculovirus-Insect cells
Species
Human
Sequence Information
A DNA sequence encoding the human SPG3A (NP_056999.2) (Met 1-Thr 447) was fused with the GST tag at the N-terminus.
Molecule Mass
The recombinant human SPG3A/GST chimera consists of 671 amino acids and and has a calculated molecular mass of 77 kDa. It migrates as an approximately 66 kDa band in SDS-PAGE under reducing conditions.

Human SPG3A Baculovirus-Insect cells Overexpression Lysate: Usage Guide

Preparation Method
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Lysis Buffer
Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.
Recommend Usage
1.  Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2.  Re-dissolve the pellet using 200μL pure water and boil for 2-5 min.
Sample Buffer
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.

Human SPG3A Baculovirus-Insect cells Overexpression Lysate: Alternative Names

Human AD-FSP Overexpression Lysate; Human atlastin1 Overexpression Lysate; Human FSP1 Overexpression Lysate; Human GBP3 Overexpression Lysate; Human HSN1D Overexpression Lysate; Human SPG3 Overexpression Lysate; Human SPG3A Overexpression Lysate

SPG3A Background Information

Atlastin-1, also known as Spastic paraplegia 3 protein A, Guanine nucleotide-binding protein 3, GTP-binding protein 3, GBP3, ATL1 and SPG3A, is a multi-pass membrane protein which belongs to theGBP family and atlastin subfamily. ATL1 / SPG3A is expressed predominantly in the adult and fetal central nervous system. Expression of ATL1 / SPG3A in adult brain is at least 5-fold higher than in other tissues. ATL1 / SPG3A is detected predominantly in pyramidal neurons in the cerebral cortex and the hippocampus of the brain. ATL1 / SPG3A is also expressed in upper and lower motor neurons (at protein level). A distinguishing feature of ATL1 / SPG3A is its frequent early onset, raising the possibility that developmental abnormalities may be involved in its pathogenesis. Missense SPG3A mutant atlastin-1 proteins have impaired GTPase activity and may act in a dominant-negative, loss-of-function manner by forming mixed oligomers with wild-type atlastin-1. Defects in ATL1 / SPG3A are the cause of spastic paraplegia autosomal dominant type 3 (SPG3), also known as Strumpell-Lorrain syndrome. Spastic paraplegia is a degenerative spinal cord disorder characterized by a slow, gradual, progressive weakness and spasticity of the lower limbs.
Full Name
atlastin GTPase 1
References
  • Zhao X., et al.,(2001), Mutations in a newly identified GTPase gene cause autosomal dominant hereditary spastic paraplegia. Nat. Genet. 29:326-331.
  • Luan Z., et al., (2002), A novel GTP-binding protein hGBP3 interacts with NIK/HGK.FEBS Lett. 530:233-238.
  • Ota T., et al.,(2004), Complete sequencing and characterization of 21,243 full-length human cDNAs.Nat. Genet. 36:40-45.
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