This Human IRAK4 overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of IRAK4 protein (Cat: 10735-H07B) from the overexpression lysate was verified.
A DNA sequence encoding the full length of human IRAK4 (Q9NWZ3-1) (Met 1-Ser 460) was expressed with a polyhistidine tag at the N-terminus.
The recombinant human IRAK4 consists of 478 amino acids and predicts a molecular mass of 53.8 kDa. It migrates as an approximately 48 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.
Human IPD1 Overexpression Lysate;Human IRAK-4 Overexpression Lysate;Human NY-REN-64 Overexpression Lysate;Human REN64 Overexpression Lysate
Interleukin-1 receptor-associated kinase 4, also known as Renal carcinoma antigen NY-REN-64, IRAK-4 and IRAK4, is a member of the protein kinase superfamily, TKL Ser/Thr protein kinase family and Pelle subfamily. IRAK4 contains one death domain and one protein kinase domain. IRAK4 is required for the efficient recruitment of IRAK1 to the IL-1 receptor complex following IL-1 engagement, triggering intracellular signaling cascades leading to transcriptional up-regulation and mRNA stabilization. It also phosphorylates IRAK1. A member of the IL-1 receptor (IL-1R)-associated kinase (IRAK) family, IRAK4, has been shown to play an essential role in Toll-like receptor (TLR)-mediated signaling. IL-1-mediated IRAK4 kinase activity in T cells is essential for induction of IL-23R expression, Th17 differentiation, and autoimmune disease. Pharmacological blocking of IRAK4 kinase activity will retain some levels of host defence, while reducing the levels and duration of inflammatory responses, which should provide beneficial therapies for sepsis and chronic inflammatory diseases. Defects in IRAK4 are the cause of recurrent isolated invasive pneumococcal disease type 1 (IPD1) which is defined as two episodes of IPD occurring at least 1 month apart, whether caused by the same or different serotypes or strains. Recurrent IPD occurs in at least 2% of patients in most series, making IPD the most important known risk factor for subsequent IPD. Defects in IRAK4 are also the cause of IRAK4 deficiency which causes extracellular pyogenic bacterial and fungal infections in otherwise healthy children.
Strelow,A. et al., 2003, FEBS Lett. 547 (1-3):157-61. Kim,T.W. et al., 2007, J Exp Med. 204 (5):1025-36. Trumstedt,C. et al., 2007, J Leukoc Biol. 81 (6):1591-8. Li,X. et al., 2008, Eur J Immunol. 38 (3):614-8. Staschke,K.A. et al., 2009, J Immunol. 183 (1): 568-77.