This Human eIF3K overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of eIF3K protein (Cat: 14553-H20B) from the overexpression lysate was verified.
A DNA sequence encoding the human EIF3K (Q9UBQ5) (Ala2-Gln218) was expressed the N-terminal polyhistidine-tagged GST tag at the N-terminus.
The secreted recombinant human EIF3K consists of 454 amino acids and predicts a molecular mass of 52.8 KDa. The apparent molecular mass of the protein is approximately 47 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.
Human ARG134 Overexpression Lysate;Human EIF3-p28 Overexpression Lysate;Human EIF3S12 Overexpression Lysate;Human HSPC029 Overexpression Lysate;Human M9 Overexpression Lysate;Human MSTP001 Overexpression Lysate;Human PLAC-24 Overexpression Lysate;Human PLAC24 Overexpression Lysate;Human PRO1474 Overexpression Lysate;Human PTD001 Overexpression Lysate
EIF3K is a member of the eIF3 subunit K family. It is a component of the eukaryotic translation initiation factor 3 (eIF-3) complex, which is required for several steps in the initiation of protein synthesis. The eIF-3 complex associates with the 40S ribosome and facilitates the recruitment of eIF-1, eIF-1A, eIF-2:GTP:methionyl-tRNAi and eIF-5 to form the 43S preinitiation complex (43S PIC). It stimulates mRNA recruitment to the 43S PIC and scanning of the mRNA for AUG recognition. EIF3K is universally expressed in human tissues. It is distributed both in nucleus and cytoplasm. EIF3K is the smallest subunit of eIF3 and it interacts with a number of other subunits of eIF3 and the 40S ribosomal subunit.
Rual JF. et al., 2005, Nature. 437 (7062): 1173-8. Shen X. et al., 2004, FEBS Lett. 573 (1-3): 139-46. Wei Z. et al., 2004, J Biol Chem. 279 (33): 34983-90.