This Rat COL4A3 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of COL4A3 protein (Cat: 80439-R01H) from the overexpression lysate was verified.
A DNA sequence encoding the rat COL4A3 (NP_001129231) (Gly1426-His1670) was expressed with Fc region of human IgG1 at the N-terminus.
The recombinant rat COL4A3 comprises 505 amino acids and predicts a molecular mass of 55.2 kDa. The apparent molecular mass of the recombinant protein is approximately 59 kDa in SDS-PAGE under reducing conditions due to glycosylation.
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).
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.
COL4A3 is a major structural component of basement membranes. It is composed of 3 alpha subunits, which are encoded by 6 different genes, alpha 1 through alpha 6. Each of these alpha subunits can form a triple helix structure with 2 other subunits to form COL4A3. Autoantibodies bind to the collagen molecules in the basement membranes of alveoli and glomeruli can cause goodpasture syndrome. COL4A3 is also linked to an autosomal recessive form of alport syndrome. COL4A3 is organized in a head-to-head conformation and each gene pair shares a common promoter.
Ghebrehiwet B. et al., 1992, J Leukoc Biol. 51 (6): 546-56. Maziers N. et al., 2006, Nephrol Ther. 1 (2): 90-100. Rana K. et al., 2005, Semin Nephrol. 25 (3): 163-70.