Redox Metabolism Shared Resource Facility Forms and Services
Before the RM SRF can begin work, researchers are required to complete and submit the RM SRF Sample Submission Form and the RM SRF Biosafety Questionnaire. Both forms are available via iLab, and may be accessed by clicking here
https://ukmcc.ilabsolutions.com/account/login. Completed forms should be sent to Dr. Mihail Mitov or Mr. Michael Alstott via iLAB for scheduling.
NOTE: RM SRF services cannot be initiated without a
researcher account number and authorization within the iLab submission form.
Researchers interested in using proteomics should contact Dr. Haining Zhu directly at
email@example.com or 859-323-3643.
Researchers interested in using metabolomics should begin by preparing to discuss questions such as: what is the problem; what is the experimental system and why; what information is already available; and whether Seahorse analyses are appropriate to conduct?
Once a researcher has answers to these questions, it is then advised to contact one of the four CESB directors listed below to begin consultation. Like all components of the RM SRF, the metabolomics component operates on a charge-back basis. It is very common for data analysis to take 90% of metabolomics labor,
so MCC researchers should be prepared to discuss time frame and cost structure with this consideration.
Request RM SRF Services
The RM SRF now uses an online program called iLab to manage service requests and billing. To start using RM SRF services, click the link below, which will take you to a landing page with more detailed instructions, including a one-time account setup. Once
your account is set up, iLab will enable you to place RM SRF service requests, provide the required approvals, and monitor the progress of your project.
RM SRF Main Services (fee for service basis)
1: Analysis of markers of oxidative and nitrosative stress. Assays of total protein oxidation and lipid peroxidation ($15/per oxidative modification/per membrane used).
- 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
2: Molecular biological manipulation of biological systems with which to investigate redox signals, including measurements of mitochondrial function. Seahorse analysis ($35/sample). Molecular biological manipulation of redox signaling ($50/sample). Assay of redox enzymes
- 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.
3: Proteomics identification of proteins. Proteomics or redox proteomics analyses of protein expression or oxidatively or covalently modified proteins. The MCC subsidizes proteomics services for MCC members at a rate of 40%. For instance, LC-MS/MS analysis of protein modifications using Orbitrap is
$90 for MCC members instead of $150. To comply with the University policy that all users should be charged with the same rate, MCC provides a $60 subsidy for this analysis. The commitment from MCC will enable the investigators to utilize the state-of-the-art technology in their cancer research programs.
- 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
- 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.
Service 4: Metabolomics – New service started in 2015. Two broad categories of metabolomics services are performed: “profiling” and Stable Isotope Resolved Metabolomics (SIRM). Profiling refers to targeted or untargeted experimental designs to determine the amounts of features in analytical
platforms (e.g. different types of MS or NMR) or the identities and amounts of compounds in samples. SIRM enables simultaneous quantitative analysis of many metabolic "pathways" and fluxes that are of especial relevance in the context of metabolic reprogramming that occurs in cancer, including energy production,
anabolic pathways necessary for proliferation, and survival pathways including oxidative stress metabolism.
A detailed list of metabolomics services and rates can be found at
Ancillary services (complimentary)
In addition, the RM SRF offers the following ancillary services:
- Education of MCC investigators on ways to prevent artifactual results and, consequently, obtain reliable and precise data. Please schedule appointments with Dr. Mitov via iLab by clicking here. [
- Writing technical descriptions, result sections and discussion paragraphs for posters, papers and grants. 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, proteomics and metabolomics.
- 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 RM SRF and FCCS SRF; additional methods involving tools of FCCS SRF for protein oxidation.
Please remember to
acknowledge the RM 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.) using the following verbiage:
This research was supported by the Redox Metabolism Shared Resource Facility Services of the University of Kentucky Markey Cancer Center (P30CA177558).
Technologies used by the RM SRF »