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://my.ilabsolutions.com/service_center/show_external/3590. 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 firstname.lastname@example.org 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.
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).
of protein oxidation (protein carbonyls and 3-nitrotyrosine).
of lipid peroxidation (protein-bound 4-hydroxy-2-trans-nonenal, HNE).
of DNA or RNA oxidation (8-hydroxy-2 deoxy-guanosine or 8-hydroxy-2-guanosine,
of antioxidant enzyme activities and levels.
of reduced and oxidized glutathione (GSH/GSSG) and NAD+/NADH,
of results and suggestions for use of these indices in the four MCC research
2: Molecular biological manipulation of biological systems
with which to investigate redox signals, including measurements of
mitochondrial function. Seahorse analysis ($40/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
- 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
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
- 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
of results in terms of pathways and functions modulated by protein oxidation.
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 http://bioinformatics.cesb.uky.edu/bin/view/RCSIRM/ServicesAndRates
Ancillary services (complimentary)
In addition, the RM SRF
offers the following ancillary services:
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. [https://my.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.
of templates for protocols of indices of oxidative stress, Seahorse technology,
proteomics and metabolomics.
of new applications of expression and redox proteomics to identify other
protein post-translational modifications at the cancer interface (e.g.,
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
Technologies used by the RM SRF »