Determination of Protein Concentration by ELISA
As part of a research project funded by the National Science Foundation, Innovabio has determined the concentration of protein X in cell culture media by sandwich ELISA. Protein X is a secreted eukaryotic protein. The ELISA results indicate that, in cell culture, the concentration of protein X depends on cell type and growth conditions. A commercially-available ELISA kit was employed to determine protein X concentration.
Kinetic analysis of a bireactant, recombinant enzyme produced and purified to homogeneity at Innovabio. The enzyme shows the classic Michaelis-Menten behavior for bireactant systems. Panel A shows product formation versus reaction time at varying concentrations of substrate 1 or 2, keeping the concentration of cosubstrate fixed. Panel B shows reaction rates plotted against varying concentrations of substrates 1 or 2 (cosubstrate concentration fixed). The Michaelis-Menten constant (Km) value for substrate 1 is 60 μM whereas for substrate 2 is 42 μM.
Gene Expression SDS-PAGE and Western
A local biotechnology company contracted Innovabio to clone and express the gene that codes for a protein with high market value. The recombinant protein was successfully purified and the protocols and materials developed in this project were transferred to the partner company. Shown above is 15% SDS-PAGE (A) and Western Blot (B) analysis of E. coli BL21 (DE3) pLysS cells transformed with expression plasmid X and induced to express gene X. Lanes: 1) Broad-range protein molecular weight markers (in kDa); 2) histidine-tagged Y protein (positive control); 3) whole-cell homogenate from un-induced cells; 4) whole-cell homogenate after 2 h induction with 0.75mM IPTG; and 5) whole-cell homogenate after 4 h induction with 0.75mM IPTG. An anti-histidine polyclonal antibody was used as a primary antibody. Histidine-tagged X and Y proteins were detected using a secondary
antibody that reacted with the NBT/BCIP reagent.
Protein Production Data Sheet
A product data sheet for one of a number of soluble and insoluble recombinant proteins Innovabio was contracted to purify to homogeneity. Innovabio developed protocols that optimized large-scale recombinant protein production as well as protocols to obtain highly pure, soluble and insoluble protein species. Soluble protein species were purified via IMAC coupled to an AKTA FPLC. Insoluble protein species were denatured with urea, refolded by way of a urea reverse gradient, and obtained as a highly pure preparation employing the aforementioned chromatographic technique and equipment.
Innovabio has conducted real-time PCR gene expression analysis on model plants to investigate the effects of several stimulants on the expression of a group of marker genes. These stimulants are currently being developed by a SLC-based company. Real-time PCR traces for expression analysis of target gene (red) and control gene (blue) are shown on panel A. The figure displays the target gene Ct values obtained for each of the experimental treatments analyzed in this work. Panel B shows dissociation curve derivative for target (red) and control (blue) gene amplicons produced after the real-time PCR runs.