Protein analysis in tissue lysates
In this example, protein lysates were made from kidney tissue and measured for a range of phosphoproteins. Actin was used as a housekeeping gene. Blots were scanned and quantified using the Licor Scanner.
Compound profiling in a mechanism of Action Study
Protein lysates were made from cells treated with compounds at various doses. Inhibition of phosphorylation was quantified by measuring signal intensity from Licor scans and normalizing to a housekeeping gene, such as Actin.
target protein analysis via western blot
Protein was isolated from tissue lysates and total protein was quantified. Equal amount of protein were loaded on SDS gels for Western blotting. Proteins of interest are probed with a housekeeping gene using IR dyes. Blots are scanned and quantified using the Licor scanner. In this example, monkey retinoid and choroid tissues were analyzed. We have experience with a range of tissues, including brain, skin, kidney, liver, and more.
IN cell western blotting
In this assay, cells are plated in a 96 well format and treated with compounds at a range of concentrations. After an established amount of time, cells are fixed. Primary antibodies for the protein of interest and a housekeeping gene are added to each well. After overnight incubation, the primary antibodies are washed away and IR-labeled secondary antibodies are added. Plates are imaged and signal intensity is quantified using the Licor Scanner.
Recombinant Protein Production
The recombinant protein of interest was expressed in HEK293 cells. The protein was deglycosylated using a PNGase Glycan cleavage kit. The de-glycosylated protein was de-tagged using HRV3C protease. The tag and enzyme were removed over a Ni-NTA column. Purified protein was dialyzed against TBS and analyzed over a 4-12% PAGE gel. The gel was stained with Coomassie stain to visualize the bands.
Antibody production
This antibody was purified from a client supplied hybridoma, Once collected from the supernatant, the antibody was passed through a MabSelect column and dialyzed. The antibody was run on a 4-20% Bis-Tris denaturing gel and stained with Coomassie.
In addition to purification of antibodies from hybridomas, antibodies can also be produced in CHO cells, through transfection of plasmids containing the heavy and light chains.
Gene Expression analysis
RNA was extracted from A549 cells post treatment and expression of a range of genes was measured via real-time QPCR.
RNA can be extracted from cells that were treated in house, at the client site, or from tissues provided by the client. RNA can also be extracted at the same time as proteins, allowing for the comparison of gene and protein expression.
GLP Assay Development
Our lab is equipped with a Good Lab Practices (GLP) room. All the work done here follows standard operating protocols and the data provided is certified as required by the FDA. Here, we can provide ELISA and MSD assay development for PK or biomarker measurement.
Compound profiling in a cell proliferation assay
For this assay, cells of interest were plated in a 96 well plate, as needed. Compounds were added to the cells in duplicate and at a range of concentrations. After 72hrs, we used Promega's CTG assay to measure % inhibition of cell growth. In this manner, we can read dozens of compounds to measure inhibition of cell proliferation by compounds of interest. In a similar manner, cell death and apoptosis can be measured using other commercially available kits. Below is a representative graph of the type of data that can be generated through this type of assay.
Differentiated Cells Visualized under a microscope
Cells were differentiated following standard cell culture protocol. As requested by the client, cells were visualized under the microscope for morphological changes prior to further functional analysis. The lab has fluorescent and brightfield microscope capabilities, allowing for the imaging of cell morphology or the quantitation of fluorescent markers.