00062 - Applied Biochemistry

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 (3 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.

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.

Applied biochemistry-3 (2 credits)-Prof. Anna Maria Ghelli

PROGRAM OF THE LABORATORY MODULE OF APPLIED BIOCHEMISTRY

At the end of the laboratory course for Applied biochemistry, the student will have learned some biochemical techniques for the study of proteins and acquired the knowledge on the principles on which they are based.
The laboratory will be structured in six different exercises, some interconnected with each other, the basic principles associated with the techniques that will be used will be explained before each pratical experience.

• COLORIMETRIC DOSAGE OF PROTEINS WITH THE BRADFORD METHOD
The purpose of this laboratory is the determination of the protein concentration of a mixture of proteins (Lactic dehydrogenase, LDH and cytochrome c, CYTC) using the Bradford colorimetric method. The result obtained will be used to determine the volume of protein mixture to be loaded on the chromatographic column in order to separate a known quantity of protein mixture.
Techniques used: UV-VIS spectroscopy

• SEPARATION OF PROTEINS BY ION EXCHANGE CHROMATOGRAPHY
The purpose of this laboratory is the separation of the two proteins from the protein mixture whose concentration was determined in the previous experience using an ion exchange chromatographic column, using their different isoelectric point. The chromatographic fractions of the two separated proteins will be used in the subsequent experiments to determine their degree of purity and their molecular weight by electrophoresis on acrylamide gel under denaturing conditions (SDS-PAGE) and to determine some kinetic parameters of lactic dehydrogenase.
Techniques used: Ion exchange chromatography, UV-VIS spectroscopy

• SEPARATION OF PROTEINS BY GEL-ELECTROPHORESIS (SDS-PAGE)
The purpose of this laboratory is to verify the successful purification of the separated proteins by ion exchange chromatography and to determine their molecular weight by means of polyacrylamide gel electrophoresis (SDS-PAGE).
Techniques used: Gel electrophoresis

• DETERMINATION OF THE Km and Vmax OF THE LACTIC DEHYDROGENASE FOR PIRUVATE AND NADH
The purpose of this laboratory is to determine some kinetic parameters of the lactate dehydrogenase that has been previously purified by ion exchange chromatography from the protein mixture. The reaction rates at different concentrations of the two substrates will be measured using a direct enzyme assay.
Techniques used: UV-VIS spectroscopy

• DETERMINATION OF THE ACTIVITY OF LACTIC DEHYDROGENASE RELEASED IN THE CELL CULTURE MEDIUM
Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that
is found in all cells. LDH is rapidly released into the cell culture supernatant when the plasma membrane is damaged. The purpose of this laboratory is to measure the release of the lactate dehydrogenase in the cell culture medium following treatment with cytotoxic substances. The presence of the enzyme in the culture medium indicates necrotic death while its absence indicates apoptotic death.
Techniques used: UV-VIS spectroscopy

• CELL VITALITY DETERMINATION ASSAY
The aim of this laboratory is to determine the number of viable cells after treatment with apoptotic and necrotic cell death inducers . The cell viability assay will done using sulforodamine B (SRB).
Detection method: Colorimetric
Platform: Microplate reader

Readings/Bibliography

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

K.Wilson e J.Walker - Biochimica e Biologia Molecolare: Principi e tecniche – NUOVA EDIZIONE (VIII) 2019

M.Maccarrone - Metodologie biochimiche e biomolecolari. Strumenti e tecniche per il laboratorio del nuovo millennio. Zanichelli 2019

Teaching methods

The course includes 6 frontal credits and 2 laboratory credits.

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.

In the 2 laboratory credits problems such as: determination of the protein content of a solution, determination of enzymatic kinetic parameters, cell viability measurements will be addressed from a practical point of view.

Assessment methods

Modules 1 and 2: 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.

Laboratory Module (module 3) : the final exam will consist in a test with 10 Quizzes/short answer questions

The The overall rating is a weighted average of the two tests.

Teaching tools

Video, PC, Overhead Projector

Office hours

See the website of Romana Fato

See the website of Cecilia Prata

See the website of Anna Maria Ghelli