FRBC Services, Technologies and Forms
The FRBC SRF provides four primary service categories and two ancillary services with a range of individual investigator services. In addition to immunochemical or high performance liquid chromatography (HPLC) determination of indices of oxidative stress, fluorescence-based screenings using microtiter plate readers and FACS analysis in cooperation with the Flow Cytometry and Cell Sorting Shared Resource Facility (FCCS SRF) can be employed to determine cellular reactive oxygen species (ROS) levels with specific ROS scavengers. As an adjunct to the specific service offering, FRBC SRF personnel also assist in interpretation of results and suggestions for use of these indices by investigators in the four MCC research programs.
Before the FRBC SRF can begin work, researchers are required to complete and submit the FRBC SRF Sample Submission Form and the FRBC SRF Biosafety Questionnaire to Dr. Mihail Mitov. NOTE: The Sample Submission Form is not complete without a researcher account number and authorization.
Please remember to acknowledge the FRBC SRF when submitting publications or giving presentations using the following statement. (Please also consider including the names of individuals from the shared resources if they provided any intellectual input or additional effort.)
- This research was supported by the Free Radical Biology in Cancer Shared Resource of the University of Kentucky Markey Cancer Center (P30CA177558).
FRBC SRF Services
Analysis of oxidative and nitrosative stress.
- Indices of protein oxidation (protein carbonyls and 3-nitrotyrosine).
- Index of lipid peroxidation (protein-bound 4-hydroxy-2-trans-nonenal, HNE).
- Index of DNA or RNA oxidation (8-hydroxy-2 deoxy-guanosine or 8-hydroxy-2-guanosine, respectively).
- Analyses of antioxidant enzyme activities and levels.
- Analyses of reduced and oxidized glutathione (GSH/GSSG) and NAD+/NADH, NADP+/NADPH.
- Interpretation of results and suggestions for use of these indices in the four MCC research programs
Redox biological analyses.
- Seahorse Biosciences instrumental analyses to monitor changes on oxygen consumption and pH in intact cells simultaneously using a microtiter plate platform (for example, to facilitate dose-response studies of chemotherapeutics).
- cDNA probes coding for primary antioxidant enzymes.
- Stable and transient transfection of redox-related proteins (including those that regulate the redox status of cells, scavenge free radicals, and repair oxidative/nitrosative damage) into cells.
- Proteomics identification of proteins with differential expression, deferential oxidative modification or differential covalent modification in systems of interest: protein separation, digestion, and ESI-MS/MS sequence analysis of tryptic peptides on an orbitrap MS instrument.
- Imaging software-mediated determination of proteins to be evaluated.
- Spot excising and protein digestion.
- Database interrogation to identify proteins.
- Validation of identification by Western blotting or other means.
- Functional analysis of oxidatively modified proteins (can also be performed for other post-translational modifications).
- Analysis of protein-protein interactions involved in redox signaling (can also be applied to other signaling pathways).
- Interpretation of results in terms of pathways and functions modulated by protein oxidation.
Electron paramagnetic resonance (EPR) detection of free radicals (new service; not yet used by MCC investigators).
- State-of-the-art equipment for detection and quantify of free radicals, antioxidants, and pro-oxidants.
- Interpretation of results.
- EPR is the only technique that can directly detect and quantitate free radicals, allowing MCC investigators to know immediately whether free radicals are involved in the systems under investigation.
In addition, the FRBC SRF offers the following ancillary services:
- Consultation on how to perform, analyze, and interpret redox chemistry and biology experiments, including oxidative/nitrosative stress damage, expression proteomics, redox proteomics, and proteomics of covalently modified proteins, mitochondrial energetics, free radical detection, antioxidant enzyme construct and development, enzyme activity assays, and thiol analyses.
- Education of MCC investigators on ways to prevent artifactual results and, consequently, obtain reliable and precise data.
- Assistance to investigators on grant proposals and manuscripts by providing technical information or preliminary data.
- Provision of templates for protocols of indices of oxidative stress, Seahorse technology, and proteomics.
Development of new applications/methods
- Pursuit of new applications of expression and redox proteomics to identify other protein post-translational modifications at the cancer interface (e.g., methylation, acetylation).
- Cross-validation studies of analyses conducted by FRBC SRF and FCCS SRF; additional methods involving tools of FCCS SRF for protein oxidation.
- Use of Seahorse Biosciences technology to gain new insights into mitochondrial biology in cancer.
- Dissemination of the availability of these new applications to MCC investigators for use in better understanding free radicals and cellular energetics in cancer and cancer therapy.
FRBC SRF Sample Submission Form
FRBC SRF Biosafety Questionnaire
Note: Before the FRBC SRF can begin work, researchers are required to complete and submit the Biosafety Questionnaire and the FRBC Sample Submission Form to Dr. Mihail Mitov.
Measures of oxidative/nitrosative stress
The FRBC SRF offers highly sensitive (ng of protein) immunochemical analyses of total oxidative and/or nitrosative stress. Dr. D. Allan Butterfield, FRBC SRF Director, has published extensively on validating this method for assessment of global oxidative stress. If desired by MCC investigators, redox-coupled reduced glutathione and oxidized glutathione are determined by fluorescence or HPLC methods, and NAD(P)H and NAD(P)+ are determined by HPLC with electrochemical detection. In the rare event that equivocal results are obtained, MS-based detection of protein carbonyls will be employed to validate the immunochemical methods or LC-MS will be used for analysis of small molecule markers of cellular redox state.
Several indices of mitochondrial changes that occur in conditions of oxidative/nitrosative stress are carried out using state-of-the-art Seahorse technology. The Warburg effect is a preponderance of energy production by glycolysis rather than through the mitochondrial electron transport chain in cancer cells. It can easily be determined on intact cells using the 96-well Seahorse instrument to determine cellular sequelae of oxidative/nitrosative stress. This platform allows dose-response studies to be carried out using mitochondrial biology as an endpoint.
Identification of proteins with differential expression is important to relate gene and protein concordance in cancer cells and to help identify key transcription factors that may play a role in cancer. Expertise in expression proteomics also exists in the FRBC SRF.
Redox proteomics identification of oxidized proteins
A key FRBC SRF service, redox proteomics, originated in the Butterfield laboratory. Principal redox proteomics approaches used include identification of proteins with excess protein carbonyls, 3-nitrotyrosine, or protein-bound HNE. Identification of proteins with excess nitrosylation of cysteine residues is also available. Once identified, such oxidatively modified proteins are placed into their respective molecular pathways to determine cellular sequelae of oxidatively dysfunctional proteins and provide new research of importance in cancer. Moreover, MS/MS sequence analyses of tryptic peptides used in redox proteomics permit identification of the amino acids on which the oxidative modification exists.
Proteomics to identify covalently modified proteins
Acetylation and methylation are two principal covalent modifications to proteins with significant relevance to cancer. Proteomics identification of acetylated or methylated proteins is an important tool in the FRBC SRF.
EPR detection of free radicals
Transient and reactive free radicals can be detected by spin trapping methods, in which a non-paramagnetic molecule (the trap, such as 5,5-dimethyl-pyrroline-N-oxide, DMPO) reacts with the transient free radical (the spin) to produce a stable, long-lived free radical detectable in the EPR instrument. Analysis of the resulting spectra often leads to the identity of the free radical species. In favorable cases, protein radicals can be observed directly. An added advantage of EPR over optical methods is absence of light-scattering effects. We are aware of all controls required, the free radicals trapped, and the necessary cognate spin traps needed. Additionally, newer techniques of mitochondrial-resident or fluorescence detection of spin adducts are available.