Redox Metabolism Shared Resource Facility Forms and Services
Before the RM SRF can begin work, researchers are required to complete the online RM SRF Sample Submission Form and the RM SRF Biosafety Questionnaire via iLab. Both forms may be accessed by visiting the iLab page below.
If you experience any problems trying to submit your service request to iLab, contact Dr. Mihail Mitov or Mr. Michael Alstott for troubleshooting.
NOTE: RM SRF services cannot be initiated without a
researcher account number and authorization within the iLab submission form.
Request RM SRF Services
For initial consultation, contact Dr. Mihail (Mike) Mitov, RM SRF Operations Manager, via iLab. At your initial consultation meeting, Dr. Mitov will direct you to the proper service component. The link below will take you to the RM SRF iLab 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.
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 shared resources if they provide intellectual input or additional effort.
This research was supported by the Redox Metabolism Shared Resource Facility of the University of Kentucky Markey Cancer Center (P30 CA177558).
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. 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
Guide to selecting services
At your initial consultation with Dr. Mitov, which will be arranged via iLab, we will conduct an assessment of your particular needs and which services are necessary.
If Service 1 or Service 2 is needed, Dr. Mitov will direct you to the RM SRF facility in 227 HSRB, and either he or Mr. Alstott will work with you.
If you are interested in Service 3 (proteomics), Dr. Mitov will direct you to Dr. Haining Zhu (firstname.lastname@example.org; 859-323-3643).
If you are interested in Service 4 (metabolomics), Dr. Mitov will direct you to Dr. Rick Higashi (email@example.com).
Prior to contacting Dr. Higashi, investigators should begin 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, they should 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.
Ancillary services (gratis)
In addition, the RM SRF offers the following ancillary services:
- Consulting with Dr. Mitov for assessment of services, design and interpretation of experiments involving oxidative stress and Seahorse XF analyses, directing investigators to proteomics or metabolomics components of RM SRF as needed.
- 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 at https://ukmcc.ilabsolutions.com/service_center/show_external/3590/
- 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.
Technologies used by the RM SRF »