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.

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.

The student must have passed Biochemistry (second year)

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

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

The laboratory practice are scheudle as follow:

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

Teaching

The course will consist of lecturers and lab experiences. 

Attendance

Attendance is strongly suggested 

Course books

1 Mauro Maccarone Metodologie biochimiche e biomolecolari. Strumenti e tecniche per il laboratorio del nuovo millennio.  Distribuzione esclusiva Zanichelli 2019

2 Bonaccorsi di Patti, M. C.; R. Contestabile; Di Salvo, M. L. Metodologie biochimiche. Espressione, purificazione e caratterizzazione delle proteine. istribuzione esclusiva Zanichelli 2019

3 Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology. 

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

Assessment

Oral examination and/or written test. 

Disabilità e DSA

Le studentesse e gli studenti che hanno registrato la certificazione di disabilità o la certificazione di DSA presso l'Ufficio Inclusione e diritto allo studio, possono chiedere di utilizzare le mappe concettuali (per parole chiave) durante la prova di esame.

A tal fine, è necessario inviare le mappe, due settimane prima dell’appello di esame, alla o al docente del corso, che ne verificherà la coerenza con le indicazioni delle linee guida di ateneo e potrà chiederne la modifica.

Teaching

The course will consist of lecturers and lab experiences. 

Attendance

Attendance is strongly suggested 

Course books

1 Mauro Maccarone Metodologie biochimiche e biomolecolari. Strumenti e tecniche per il laboratorio del nuovo millennio.  Distribuzione esclusiva Zanichelli 2019

2 Bonaccorsi di Patti, M. C.; R. Contestabile; Di Salvo, M. L. Metodologie biochimiche. Espressione, purificazione e caratterizzazione delle proteine. istribuzione esclusiva Zanichelli 2019

3 Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology. 

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

Assessment

Oral examination and/or written test. 

Disabilità e DSA

Le studentesse e gli studenti che hanno registrato la certificazione di disabilità o la certificazione di DSA presso l'Ufficio Inclusione e diritto allo studio, possono chiedere di utilizzare le mappe concettuali (per parole chiave) durante la prova di esame.

A tal fine, è necessario inviare le mappe, due settimane prima dell’appello di esame, alla o al docente del corso, che ne verificherà la coerenza con le indicazioni delle linee guida di ateneo e potrà chiederne la modifica.