- Docente: Romana Fato
- Credits: 8
- SSD: BIO/10
- Language: Italian
- Moduli: Cecilia Prata (Modulo 1) Romana Fato (Modulo 2)
- Teaching Mode: Traditional lectures (Modulo 1) Traditional lectures (Modulo 2)
- Campus: Bologna
- Corso: Single cycle degree programme (LMCU) in Chemistry and Pharmaceutical Technologies (cod. 8412)
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