Amino acid analysis is performed using the old Hewlett Packard AminoQuant method adapted to a newer system.  The system consists of a Agilent 1260  liquid chromatograph that is equipped with a programmable autosampler for automated sample handling, derivatization and injection, a variable wavelength UV detector for low-moderate sensitivity (Agilent G1365D) analyses and an in-line Agilent G1321B fluorescence detector for high-sensitivity analyses (fluorescent detection).  All system control and data analysis is performed by Agilent Chemstation software.  Liquid and vapor phase hydrolysis protocols have been developed in order to service a wide range of sample types (liquid, dried and electroblotted proteins on membranes).  We offer routine validated (qualitative and quantitative) amino acid compositions of purified proteins and synthetic peptides as well as (quantitative ) free-amino acid (or total amino acid) compositions of feeds, flours and physiological fluids.  Hydrolyzed proteins samples and free amino acids are derivatized pre-column with o-phthalaldehyde (OPA) and 9-fluoromethyl-chloroformate (FMOC) prior to separation and quantitation by reverse phase HPLC.  Sensitivity is in the picomole to nanomole range depending upon sample matrix contributions.

Electrophoresis has been a valuable method of protein analysis for decades.  In recent years, the increased sensitivity of automated sequencers and analyzers has enabled the use of electrophoretic separation techniques to be used as preparative methods for subsequent stages of high-sensitivity protein analysis.  The Laboratory is fully equipped to perform a variety of routine electrophoretic separations of proteins in polyacrylamide or agarose matrices. Sodium dodecyl sulfate polyacrylamide (SDS) and non-denaturing gels are available in standard and mini-gel format (linear and gradient format) as are 2-dimensional electrophoretic separations. Analytical isoelectric focusing (IEF) is performed on an Amersham Pharmacia Biotec IPGPhor electrophoresis module (first dimension) using immobilized pH gradients in polyacrylamide strips (DryStrips).  The second dimension of resolution is obtained on a vertical slab gel electrophoresis unit equipped with an external re-circulating cooling device.

Electroblotting of proteins to solid membrane supports has been used for many years in protocols used for immunodetection of antigens.  Recent developments in membrane support technology have led to the production of high-capacity, chemically inert matrices that are resistant to N-terminal sequencing chemistries.  Proteins blotted to these matrices can be excised from a mixture of proteins and subjected directly to sequence analysis or preliminary manipulations such as direct In situ chemical or proteolytic cleavage with subsequent peptide extraction and purification.  The Laboratory is equipped to perform electroblotting procedures as well as In situ  or “in gel” digestion procedures as required to obtain sequence information and protein identity.

 

 

Typhoon Trio Fluorescent Imager

The FLA 9500 Typhoon (GE Healthcare) is a high performance gel and blot imager. The imager has powerful laser excitation sources and high-quality confocal optics that allow for the sensitive detection of low-abundance targets. Red, Green and Blue excitation wavelengths (488 nm, 532 nm, 633 nm) and a wide choice of emission filters enable imaging of an extensive variety of fluorophores. The 9500 is also useful for Storage Phosphor Autoradiography, Chemiluminescence and Chemifluorescence detection. (Client must have their own Phosphor screens. The PCL does not own any.) Please call ahead with specific questions. The instrument is used on a first-come, first-served basis, but reservations are recommended for analyses that will require extensive periods of time.

Ettan Robotic Systems

The Digester is a highly versatile instrument designed to perform in-gel digestion of proteins captured in 2D gel electrophoresis spots. Default digestion protocols can be customized. The instrument performs automatic tracking of samples.

 

The Ettan Spotter is designed to automatically mix and spot digested proteins and matrix onto any MALDI sample plate.

 

 

High Pressure Liquid Chromatography

Liquid chromatography is used extensively to purify proteins and peptides from mixtures of natural materials.  The PCL has a Agilent Technologies 1260 HPLC system is equipped with an autosampler and a fluorescence detector dedicated to Amino Acid Analysis.  In addition, the facility has a GE AKTA pure chromatography system for aqueous and organic mobile phases for native protein purification as well as peptide isolation.  The AKTA pure is used to develop pilot protein purification protocols using a library of columns that includes hydrophobic interaction, ion exchange, metal chelation and affinity matrices.  In addition, the systems are used for Immuno-Depletion of contaminating proteins from physiological samples prior to Proteomic analysis.