Mouse GAD65 Baculovirus-Insect cells Overexpression Lysate


Mouse GAD65 Baculovirus-Insect cells Overexpression Lysate: 产品信息

This Mouse GAD65 overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of GAD65 protein (Cat: 50653-M20B) from the overexpression lysate was verified.
Baculovirus-Insect cells
A DNA sequence encoding full length of mouse GAD2 (NP_032104.2) (Met 1-Leu 585) was fused with the N-terminal polyhistidine-tagged GST tag at the N-terminus.
The recombinant mouse GAD2/GST chimera consists of 822 amino acids and has a calculated molecular mass of 93 kDa. It migrates as an approximately 80 kDa band in SDS-PAGE under reducing conditions.

Mouse GAD65 Baculovirus-Insect cells Overexpression Lysate: 使用指南

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.
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.
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.
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
稳定性 & 储存条件
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.
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.

Mouse GAD65 Baculovirus-Insect cells Overexpression Lysate: 别称

Mouse 6330404F12Rik Overexpression Lysate; Mouse GAD(65) Overexpression Lysate; Mouse Gad-2 Overexpression Lysate; Mouse GAD65 Overexpression Lysate

GAD65 背景信息

Glutamate decarboxylase 2, also known as glutamate decarboxylase 65 kDa isoform, 65 kDa glutamic acid decarboxylase, GAD2 and GAD65, is a member of thegroup II decarboxylase family. GAD2 is identified as a major autoantigen in insulin-dependent diabetes. GAD2 is responsible for catalyzing the production of gamma-aminobutyric acid from L-glutamic acid. A pathogenic role for this enzyme has been identified in the human pancreas since it has been identified as an autoantibody and an autoreactive T cell target in insulin-dependent diabetes. GAD2 may also play a role in the stiff man syndrome. GAD2 is implicated in the formation of the gamma-aminobutyric acid (GABA), a neurotransmitter involved in the regulation of food intake. GABA is synthesized in brain by two isoforms of glutamic acid decarboxylase (Gad), GAD1 and GAD2. GAD1 provides most of the GABA in brain, but GAD2 can be rapidly activated in times of high GABA demand. Mice lacking GAD2 are viable whereas deletion of GAD1 is lethal. Deletion of GAD2 increased ethanol palatability and intake and slightly reduced the severity of ethanol-induced withdrawal.
glutamate decarboxylase 2 (pancreatic islets and brain, 65kDa)
  • Karlsen A.E., et al.,(1991), Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10. Proc. Natl. Acad. Sci. U.S.A. 88:8337-8341.
  • Bu D.-F., et al., (1992), Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene.Proc. Natl. Acad. Sci. U.S.A. 89:2115-2119.
  • Bu D.-F., et al.,(1994), The exon-intron organization of the genes (GAD1 and GAD2) encoding two human glutamate decarboxylases (GAD67 and GAD65) suggests that they derive from a common ancestral GAD.Genomics 21:222-228.
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