This Human CDK7 & CCNH & MNAT1 overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of CDK7 & CCNH & MNAT1 protein (Cat: CT020-H07B) from the overexpression lysate was verified.
A DNA sequence encoding the human CDK7 (P50613) (Ala 2-Phe 346) was fused with a polyhistidine tag at the N-terminus, constructed the plasmid 1; A DNA sequence encoding the human CCNH (P51946) (Tyr 2-Leu 323) was fused with a polyhistidine tag at the N-terminus, constructed the plasmid 2. A DNA sequence encoding the human MNAT1 (P51948) (Asp 2-Ser 309) was fused with a polyhistidine tag at the N-terminus, constructed the plasmid 3. The three plasmids were co-expressed and the heterotrimer was purified.
The recombinant heterotrimer of human CDK7/CCNH/MNAT1 comprises 1032 (364 + 341 + 327) amino acids and has a calculated molecular mass of 118.8 (41.2 + 39.7 + 37.9) kDa. The apparent molecular mass of rh CDK7/CCNH/MNAT1 heterotrimer is approximately 25,38 & 44 kDa respectively 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.
Human CAK1 Overexpression Lysate; Human CDKN7 Overexpression Lysate; Human HCAK Overexpression Lysate; Human MO15 Overexpression Lysate; Human p39MO15 Overexpression Lysate; Human STK1 Overexpression Lysate