Biochemistry and analytical biochemistry - BIOCHIMICA E BIOCHIMICA ANALITICA: BIOCHIMICA ANALITICA (2016/2017)

Course code
4S02696
Credits
4
Other available courses
Other available courses
    Academic sector
    BIO/10 - BIOCHEMISTRY
    Language of instruction
    Italian
    Teaching is organised as follows:
    Activity Credits Period Academic staff Timetable
    teoria 2 I sem. Alessandra Astegno
    laboratorio [1° turno] 2 I sem. Alessandra Astegno
    laboratorio [2° turno] 2 I sem. Alessandra Astegno

    Lesson timetable

    I sem.
    Activity Day Time Type Place Note
    teoria Tuesday 10:30 AM - 12:30 PM lesson Lecture Hall H from Oct 18, 2016  to Jan 31, 2017
    laboratorio [1° turno] Wednesday 1:30 PM - 6:30 PM laboratorio Laboratory Laboratorio di Biochimica from Oct 26, 2016  to Jan 31, 2017
    laboratorio [2° turno] Thursday 2:30 PM - 7:30 PM laboratorio Laboratory Laboratorio di Biochimica from Oct 27, 2016  to Jan 31, 2017

    Learning outcomes

    The course is designed to provide the basic knowledge of the main biochemical methods used in the separation, identification, characterization and analysis of biomolecules, with particular reference to applied strategies used for the study of proteins.

    Syllabus

    Protein purification
    pH-buffer solutions: choice of buffers for biochemistry. Amino acids and isoelectric point. Homogenization: choice of methods and media. 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. Purification of enzymes: specific activity and yield.
    Spectroscopic techniques
    UV-VIS in Biochemistry. Report of Lambert-Beer. Colorimetric assays. Spectrofluorimetry (general principles and applications) and Chemiluminescence. Fluorescent proteins. Fluorescence resonance energy transfer (FRET).
    Centrifugal techniques
    Principles of sedimentation, RCF, sedimentation coefficient and sedimentation times. Centrifuges and rotors. Differential centrifugation: separation and analysis of subcellular fractions. Density gradient centrifugation, zonal and isopycnic. Applications: separation of cells, subcellular organelles, proteins, nucleic acids.
    Chromatographic techniques
    General principles, resolution and theoretical plates. Column chromatography. Hydrophobic chromatography of proteins (HIC).Ion-exchange chromatography of proteins. Exclusion chromatography (gel filtration). Affinity chromatography. Immuno-affinity chromatography. Chromatography with dyes and lectins. Chromatography with metals; purification of histidine-tagged proteins and of recombinant proteins.
    Electrophoretic techniques
    General principles, factors that influence the electrophoretic mobility. Gel Electrophoresis: PAGE, SDS-PAGE, Agarose. Methods of detection and quantitative assessments. Blotting -Western blotting in the study of proteins. Isoelectric focusing (IEF). Capillary electrophoresis. Two-dimensional electrophoresis.
    Practical laboratory excercises
    1. Determination of protein concentration by absorbance at 280 nm a nd by colorimetric assay.
    2. Determination of the NADH absorbance UV-Vis spectrum and determination of the extinction coefficient of NADPH
    3. Determination of kinetic parameters (vmax and Km) of acid phosphatase
    4. Determination of protein molecular weight by size-exclusion chromatography
    5. Gel Electrophoresis SDS-PAGE
    6. Western blotting
    7. Oral presentation of a scientific journal paper

    Assessment methods and criteria


    The exam consists of a written examination and a facultative oral examination

    Reference books
    Activity Author Title Publisher Year ISBN Note
    teoria Monica Stoppini, Vittorio Bellotti Biochimica applicata EDISES 2012
    laboratorio M. C. Bonaccorsi di Patti, R. Contestabile, M. L. Di Salvo Metodologie Biochimiche Casa Editrice Ambrosiana 2012
    laboratorio Keith Wilson, John Walker-Edizione italiana a cura di M.S. Pilone e L. Pollegioni Biochimica e biologia molecolare - Principi e tecniche - Le bioconoscenze e le biotecnologie in laboratorio Raffaello Cortina Editore 2006

    STUDENT MODULE EVALUATION - 2016/2017