Università degli Studi di Urbino Carlo Bo / Portale Web di Ateneo


BIOCHEMICAL METHODOLOGY
METODOLOGIE BIOCHIMICHE

A.Y. Credits
2019/2020 8
Lecturer Email Office hours for students
Michele Menotta

Assigned to the Degree Course

Biological Sciences (L-13)
Curriculum: SANITARIO MOLECOLARE
Date Time Classroom / Location
Date Time Classroom / Location

Learning Objectives

The aims of the course is to provide students with the tools  to understand the methods for the study of proteins (enzymes) and metabolites in animal and vegetable cells. With the lab experiences, the students will be able to assay the activity of an enzyme, to purify a protein and measure the concentration of a metabolite as well as to perform an electrophoresis.

Program

Principles of biochemical methods

Main tools/instruments of biochemical laboratories

Solutions and buffers

Centrifugation techniques. Separations of cells and subcellular organules by isopycnic and differential centrifugation

Red Blood cells lysis and preparation of the haemolysates. Buffers and antiproteolitic cocktails, reducing and stabilizing compounds.

Omogenization of tissues by means of potter, ultraturrax. French Press, sonication.

Protein studies and quantification: absorbance at 280 e 260 nm, method of Bradford. Biureto, Lowry.

Protein purification by conventional chromatography: gel filtration, Ionic exchange, affinity, immunoaffinity. Hydrophobic interactions. High performance liquid chromatography (HPLC) by riverse and direct phase.

Protein primary structure determination by means of the Edman degradation.

Electrophoresis on polyacrylamide gel under native and denaturing conditions (SDS-PAGE), densitometric analysis of the protein bands. Determination of protein molecular weight. Preparative electrophoresis, isoelectrofocusing. 2-D Electrophoresis. Capillary Electrophoresis.

Metabolite assays: deproteinization methods. Assay of metabolites by spectrophotometric. fluorimetric and radiochemical methods.

Advanced spectroscophy: Infra red spectroscopy. NMR, EPR, Mass Spectrometry.

Immunochemical techniques: production of monoclonal and plyclonal antibodies, immunotitration curves, immunocytochemical detection of proteins. Direct and indirect ELISA systems. Radiochemical methods (RIA).

Measurement of enzymatic activity: spectrophotometric. Fluorimetric and radiochemical methods. Determination of the kinetic characteristics: Km. Ki. pH curves. Thermal stability.

Bridging Courses

The student must have passed Biochemistry (second year)

Learning Achievements (Dublin Descriptors)

The student must demonstrate the understanding of the concepts and theories provided by the course. Not only is required the knowledge of the described methods, but also the chemical and physical principles behind the methods (provided during the course). Students have to demonstrate their ability to reason by the discipline logic, and have  to independently solve biochemical problems

Teaching Material

The teaching material prepared by the lecturer in addition to recommended textbooks (such as for instance slides, lecture notes, exercises, bibliography) and communications from the lecturer specific to the course can be found inside the Moodle platform › blended.uniurb.it

Supporting Activities

The laboratory practice are scheudle as follow:

Cellular lysate preparation and protein extraction. Protein Assays. SDS-PAGE. Western Blotting


Didactics, Attendance, Course Books and Assessment

Didactics

The course will consist of lecturers and lab experiences. 

Attendance

Attendance is strongly suggested 

Course books

1 Maria Carmela Bonaccorsi, Roberto Contestabile, Martino Di Salvo: Metodologie biochimiche Casa Editrice Ambrosiana. Distribuzione esclusiva Zanichelli 2012

2 Carlo De Marco, Chiara Cini: Principi di metodologia biochimica. Casa Editrice Piccin.

Assessment

Oral examination and/or written test. 

Additional Information for Non-Attending Students

Didactics

The course will consist of lecturers and lab experiences. 

Attendance

Attendance is strongly suggested 

Course books

1 Maria Carmela Bonaccorsi, Roberto Contestabile, Martino Di Salvo: Metodologie biochimiche Casa Editrice Ambrosiana. Distribuzione esclusiva Zanichelli 2012

2 Carlo De Marco, Chiara Cini: Principi di metodologia biochimica. Casa Editrice Piccin.

Assessment

Oral examination and/or written test. 

« back Last update: 17/02/20

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