This Human STAT4 overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of STAT4 protein (Cat: 13189-H07B) from the overexpression lysate was verified.
A DNA sequence encoding the human STAT4 (Q14765) (Met 1-Glu 748) was expressed, with a polyhistidine tag at the N-terminus.
The recombinant human STAT4 consists of 767 amino acids and predicts a molecular mass of 88 kDa. It migrates as an approximately 70 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.
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.
Thierfelder WE, et al. (1996) Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells. Nature. 382(6587):171-4.Hye-Soon L, et al. (2007) Association of STAT4 with Rheumatoid Arthritis in the Korean Population. Mol Med. 13(9-10): 455-60.Chitnis T, et al. (2001) Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis. J Clin Invest. 108(5): 739-47.