00062 - Applied Biochemistry

Course Unit Page

Academic Year 2018/2019

Learning outcomes

At the end of the course the student will have acquired the basic knowledge of the main biochemical methods  used in the separation, identification, characterization and analysis of biomolecules, - It will be able to design or evaluate experimental protocols used in biochemical research.

Course contents

Applied Biochemistry-1 (3 credits) - Dr. Cecilia Prata

Introduction. Principles of biochemical research. Biochemical experimentation. Introduction to Microscopy.

Cell cultures

Biological samples

 pH-buffer solutions: choice of buffers for biochemistry. Amino acids and isoelectric point.

Homogenization: choice of methods and media.

Centrifugal techniques: principles of sedimentation, RCF, sedimentation coefficient and sedimentation times. Centrifuges and rotors. Differential centrifugation: separation and analysis of subcellular fractions; differential flotation of lipoproteins. Density gradient centrifugation, zonal and isopycnic. Applications: separation of cells, subcellular organelles, proteins, nucleic acids. Evaluation of yield and enrichment, "markers" subcellular

Fractional precipitation of proteins: fractional precipitation with ammonium sulfate and isoelectric precipitation with organic solvents and polymers; heat precipitation.

Dialysis and ultrafiltration: principles and applications.

Extraction (outline)

Chromatographic techniques: general principles, resolution and theoretical plates. Column chromatography.

Adsorption chromatography. Notes on TLC: two-dimensional chromatography. Hydrophobic chromatography of proteins (HIC).

Ion-exchange chromatography of proteins. Automatic amino acid analyzer.

Exclusion chromatography (gel filtration).

Affinity chromatography. Immuno affinity. Chromatography with dyes and lectins. Chromatography with metals;  purification of histidine-tagged proteins and of recombinant proteins.

Applied Biochemistry-2 (5 credits) – Prof. Romana Fato

HPLC.

Purification of enzymes: specific activity and yield. Immobilized enzymes.

Determination of the protein sequence (Edman sequencing and mass spectrometry).

Electrophoretic techniques: general principles, factors that influence the electrophoretic mobility. Electrophoresis free phase. Zone electrophoresis on cellulose acetate, serum proteins. Gel Electrophoresis: PAGE, SDS-PAGE, Agarose. Methods of detection and quantitative assessments. Blotting - Western blotting in the study of proteins: biochemical and diagnostic applications.

Isoelectric focusing (IEF). Two-dimensional electrophoresis.

Spectroscopic techniques

UV-VIS in Biochemistry. Report of Lambert-Beer. Colorimetric assays.

Enzymatic techniques. Elements of enzyme kinetics: initial speed, effect of the concentration of enzyme and substrate, pH and temperature. Reversible and irreversible inhibition. Competitive, non-competitive and a-competitive inhibitors. Chemical modification of proteins: inhibitors as drugs. Measurement of enzyme activity: continuous methods, discontinuous, direct, indirect and coupled.

Evaluation of enzymes and metabolites plasma / serum diagnostic purposes. Types of clinical laboratory biochemical analysis. The blood sample. Enzymes present in biological materials, their dosage and clinical significance of the evaluation of the activity. General characteristics, biochemical role and methods for the determination of the main enzymes of clinical interest: LDH, CPK, AST and ALT, alkaline phosphatase, cholinesterase. Enzymatic profiles: heart, liver and pancreatic. Metabolites of clinical interest: glucose (blood sugar, glucose tolerance test, glycated hemoglobin) cholesterol and lipid disorders. Plasma proteins: nature, function, and meaning of the quantitative determination of diagnostic importance. Plasma lipoproteins: significance of quantitative determination and diagnostic significance.


Spectrofluorimetry (general principles and applications) and Chemiluminescence;  flow-cytometry and "cell sorting". Elements of luminometry.

Electrochemical techniques. Oxygen electrode; studies on mitochondrial respiration. Biosensors.

Radioisotopic methods. Principles, instrumentation and applications. Calls on the types of radioactive decay. Energy and speed of radioactive decay. Detection and measurement of radioactivity. Counting efficiency and quenching. Autoradiography. Applications of radioisotopes in biochemistry: a study of metabolic pathways, study of systems of transport and absorption, binding studies. Application of radioisotopes in clinical analysis.

Immunochemical techniques. Structure of antibodies, antigen-antibody reaction. Production of antisera, monoclonal antibodies. Methods of analysis: reaction of free phase immunoprecipitation and gel, immunodiffusion, radioimmunoassay RIA and IRMA; Enzyme immunoassays: EMIT, ELISA and PEIA. Hormone assays: methods and applications. Laboratory diagnosis of pregnancy: dose of human chorionic gonadotropin.

Molecular biology techniques. Recombinant DNA technology: general principles. Polymerase Chain Reaction (PCR): principles and diagnostic applications, forensic and paleobiological. VNTR sequences: paternity test. Identification of specific sequences of DNA and RNA: Southern hybridization (Southern blotting) and applications in the diagnosis of genetic diseases. Northern blotting and applications in the study of gene expression. Evaluation of gene expression: the technique of DNA microarray. Notes on proteomics.

Of each technique are discussed instrumental aspects and applications with related examples.

Readings/Bibliography

K.Wilson and J.Walker - Biochemistry and Molecular Biology: Principles and Techniques - Cortina Editore, 2006

Teaching methods

During the lectures will be discussed the methods connected with the trial in both biochemical and instrumental aspects of  the applications. For each technique will be presented and  discussed some practical examples.

Assessment methods

Success of learning outcomes will be assessed by an oral examination on both modules, which can not be sustained in separated times of verification.
At the end of the course the student must:
- Know the basic principles of the main biochemical methodologies used in the separation, identification, characterization and analysis of biomolecules;
- Be able to design or evaluate experimental protocols used in biochemical research.
The duration of the oral examination is on average 30-45 minutes.
The final grade for the course Applied Biochemistry (Modules I and II) is defined by an oral test on three main questions that will focus on topics related to the objectives of the course. The evaluation of the preparation of the candidate may make use of additional questions to specify aspects related to the three main questions.
Will be assessed the student's ability to be able to move within the various techniques and biochemical applications. The achievement by the student of an organic vision of the issues addressed in joint lesson critical to their use, demonstrating ownership of a mastery of expression and specific language will be assessed with a mark of excellence.

Teaching tools

Video, PC, Overhead Projector

Office hours

See the website of Romana Fato

See the website of Cecilia Prata