This Human TGF-beta 1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of TGF-beta 1 protein (Cat: 10569-H08H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain of human beta IG-H3 (NP_000349.1) precursor (Met 1-His 683) was expressed, fused with a polyhistidine tag at the C-terminus.
The secreted recombinant human beta IG-H3 comprises 671 amino acids with a predicted molecular mass of 74 kDa. As a result of glycosylation, the apparent molecular mass of rh beta IG-H3 is approximately 65 kDa 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.
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.
Human BIGH3 Overexpression Lysate;Human CDB1 Overexpression Lysate;Human CDG2 Overexpression Lysate;Human CDGG1 Overexpression Lysate;Human CSD Overexpression Lysate;Human CSD1 Overexpression Lysate;Human CSD2 Overexpression Lysate;Human CSD3 Overexpression Lysate;Human EBMD Overexpression Lysate;Human LCD1 Overexpression Lysate
TGFBI is an RGD-containing protein that binds to type I, II and IV collagens. The RGD motif is found in many extracellular matrix proteins modulating cell adhesion and serves as a ligand recognition sequence for several integrins. TGFBI plays a role in cell-collagen interactions and may be involved in endochondrial bone formation in cartilage. TGFBI is induced by transforming growth factor-beta and acts to inhibit cell adhesion. Mutations in TGFBI are associated with multiple types of corneal dystrophy. TGFBI can bind to type I, II, and IV collagens. This adhesion protein may play an important role in cell-collagen interactions. In cartilage, TGFBI may be involved in endochondral bone formation. Loss of the TGFBI is sufficient to induce specific resistance.