Human Insulin Receptor Baculovirus-Insect cells Overexpression Lysate: Product Information
This Human Insulin Receptor overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of Insulin Receptor protein (Cat: 11081-H20B1) from the overexpression lysate was verified.
A DNA sequence encoding the human INSR isoform long (NP_000199.2) cytoplasmic domain (Gly 989-Ser 1382) was fused with the N-terminal polyhistidine-tagged GST tag at the N-terminus.
The recombinant human INSR (989-1382)/GST chimera consists of 631 amino acids and has a calculated molecular mass of 72.3 KDa. It migrates as an approximately 70 KDa band in SDS-PAGE under reducing conditions.
Human Insulin Receptor Baculovirus-Insect cells Overexpression Lysate: Usage Guide
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.
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).
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.
Western Blot (WB) Optimal dilutions/concentrations should be determined by the end user.
Human Insulin Receptor Baculovirus-Insect cells Overexpression Lysate: Alternative Names
Human CD220 Overexpression Lysate; Human HHF5 Overexpression Lysate; Human Insulin Receptor Overexpression Lysate
Insulin Receptor Background Information
INSR (Insulin receptor), also known as CD22, is a transmembrane receptor that is activated by insulin. INSR belongs to theprotein kinase superfamily, and exists as a tetramer consisting of two alpha subunits and two beta subunits linked by disulfide bonds. The alpha and beta subunits are encoded by a single INSR gene, and the beta subunits pass through the cellular membrane. As the receptor for insulin with tyrosine-protein kinase activity, INSR associates with downstream mediators upon binding to insulin, including IRS1 (insulin receptor substrate 1) and phosphatidylinositol 3'-kinase (PI3K). IRS-1 binding and phosphorylation eventually leads to an increase in the high affinity glucose transporter (Glut4) molecules on the outer membrane of insulin-responsive tissues. INSR isoform long and isoform short are expressed in the peripheral nerve, kidney, liver, striated muscle, fibroblasts and skin, and is found as a hybrid receptor with IGF1R which also binds IGF1 in muscle, heart, kidney, adipose tissue, skeletal muscle, hepatoma, fibrobasts, spleen and placenta. Defects in Insulin Receptor/INSR are the cause of Rabson-Mendenhall syndrome (Mendenhall syndrome), insulin resistance (Ins resistance), leprechaunism (Donohue syndrome), and familial hyperinsulinemic hypoglycemia 5 (HHF5). It may also be associated with noninsulin-dependent diabetes mellitus (NIDDM).
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Ullrich A., et al., (1985), Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes.Nature 313:756-761.
Grimwood J., et al.,(2004), The DNA sequence and biology of human chromosome 19.Nature 428:529-535.
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