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Protein Structure Core Facility
Laurey Steinke, PhD, Director and Michele Fontaine, BS, Researcher
Dept of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198
[email protected]
402-559-6647
http://www.unmc.edu/pscf/
ABSTRACT
Simple Western by Size or Charge
ProteinSimple Peggy Sue
• Nanocapillary system
• 96 samples
• Size = Western
• Charge = isoelectric focusing (IEF)
• Can determine PTMs
The UNMC Protein Structure Core Facility (PSCF), founded in 1988, provides
information on the primary structure, identification and quantification of proteins for
the University of Nebraska System, as well as patrons outside the system.
University of Nebraska members, paying with their cost center, receive a subsidized
rate. Amino acid analysis is performed with a Hitachi L-8800. Quantitation and
amino acid composition are reported. Samples range from purified protein to spent
media, and include stream water, for determination of amino acid markers used by
spawning fish. The primary sequence of proteins is analyzed using automated
Edman degradation on a Shimadzu PPSQ 33A protein sequencer. The exact Nterminal amino acids of proteins or peptides are reported, for identification of
cleavage sites, expression start-sites, mutations, and determination of amino acid
sequence. A Waters ACQUITY UPLC H-Class BioSystem (with the ability to run
HPLC columns as well) is used for peptide and protein purification, and is available
on a per hour basis to trained patrons for analytical UPLC. Nanocapillary
electrophoresis, with separation by protein size or charge, utilizing antibody
detection is performed on the ProteinSimple PeggySue. Also called a “simple
western”, this technology allows determination of the phosphorylation state of a
protein and quantifies the amount producing publication ready figures. Visualization
of entire signaling pathways is possible in one overnight run (after optimization).
Free training on this instrument is currently available, and use is subsidized by
INBRE. Consultation with the core facility director on experimental design and grant
proposal submission is available by appointment, and is subsidized by the NRI. The
PSCF supports the research of investigators throughout the University of Nebraska
System and also receives samples from throughout the country.
Amino acid Analysis
Sample Preparation
• Lysate with compatible buffers,
concentrated.
• Antibody, will need optimization
• Secondary antibody if primary not raised
in rabbit or mouse
Edman Degradation
•
•
•
•
Analyze N-terminus of protein
Determine site of cleavage
Verify peptide sequence
Determine amino acid sequence
from non-model organisms.
• Validate sequence for FDA
requirements.
• Determine exact start site of cloned
protein expression
Gro
GroEL from thermophillic bacteria.
Auen and Steinke, unpublished data
Shimadzu PPSQ-33
• 10 picomole protein or
peptide
• PVDF
• In solution without salt or
buffer
• ProSorb available for
samples needing cleaning
IEF immunoassay of native heterotrimeric RPA
separated on a pH 5-6 gradient
Services Available
•
•
•
•
Hitachi L 8800
• 1 to 10 µgrams of protein
• 250 to 2000 picomoles of peptide
Calibration curve aspartate
Calibration curve aspartate
Quality Controls for Amino Acid Analysis
• NIST BSA hydrolyzed with each set.
• Average Error for % of each amino acid: 4.2%
• RSD for amount: 3.4%
• Seven standards are run with each sample set.
Confirmation of N-terminus of cvTBLR1
by Edman degradation analysis of the
protein, overlay plot of the first five
cycles. Color code for the chromatogram
PP
matches the colors of the identified amino
acids. From: Daniels, G….Steinke, L. ….and
P. Lee, 2015, in preparation.
Protein/Peptide Sequencing by Edman Degradation
Amino acid analysis
Simple Westerns by Size or Charge
Consultation with the director on sample preparation, experimental design and
data interpretation.
Chargeback
UPLC/HPLC
Analysis
The PSCF is a nonprofit fee-for-service facility Costs for investigators within the
University system are partially subsidized
Waters ACQUITY UPLC H-Class Biosystem
Water
• Bio-inert flow path
• Empower software
• Quaternary gradient
• Fraction Collector/Autosampler
• Dual wavelength UV/Vis Detector
• Available by the hour
Acknowledgements
The UNMC Protein Structure Core Facility receives partial support from the NEINBRE 8P20GM103427 grant (James Turpen, PI) and the Nebraska Research
Initiative.