This Mouse Smad5 overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of Smad5 protein (Cat: 50728-M20B) from the overexpression lysate was verified.
A DNA sequence encoding the mouse SMAD5 (P97454) (Thr2-Ser465) was expressed with the N-terminal polyhistidine-tagged GST tag at the N-terminus.
The recombinant mouse SMAD5/GST chimera consists of 701 amino acids and has a calculated molecular mass of 79.9 kDa. The recombinant protein migrates as an approximately 75-88 kDa band in SDS-PAGE under reducing conditions.
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.
SMAD5 is a member of the SMAD family. Members of this family mediate signal transduction by the TGF-beta/activin/BMP-2/4 cytokine superfamily from receptor Ser/Thr protein kinases at the cell surface to the nucleus. SMAD5 is involved in the TGF-beta signaling pathway that results in an inhibition of the proliferation of hematopoietic progenitor cells. It is also involved in cell signalling and modulates signals of bone morphogenetic proteins (BMP's). The binding of ligands causes the oligomerization and phosphorylation of the SMAD5 protein. SMAD5 is a receptor regulated SMAD (R-SMAD) and is activated by bone morphogenetic protein type 1 receptor kinase.
SMAD family member 5
Vinayagam A. et al., 2011, Sci Signal. 4 (189): rs8.
Sangadala S. et al., 2007, J Biomol Struct Dyn. 25 (1): 11-23.
Riggins GJ. et al., 1996, Nat Genet. 13 (3): 347-9.