Ateneo - UPO

Measuring biological things.
Techniques for sample preparation in biochemistry
Spectrophotometry.
Chromatography
Protein characterization.
Using antibodies in biotechnology.
Enzymes in diagnostic and research.
Binding assays.

"Introduzione alla biochimica di Lehninger"
David L. Nelson &Michael M. Cox
Zanichelli

The students MUST have successfully attended the "safety in chemical and biological laboratories" course.
The knowledge on the structure and properties of molecules provided by organic chemistry are preparatory.

The course includes:
- lectures on the main techniques,
- simulations using specific software for the key techniques used in biochemistry laboratories,
- practical exercises in chemical-biological teaching labs.
Attendance is compulsory. In order to obtain the laboratory grade, each student must attend at least 75% of the classes (i.e., at least 22 out of the total 30 hours of the course).

At the end of the practical sessions, each student is required to submit a laboratory notebook, compiled during the activities, documenting the procedures carried out and the understanding of the contents. A similar submission is required at the end of the “virtual” exercises performed with simulation software.
On the last day of the practical laboratory, a verification test will be administered to assess the skills acquired and the understanding of the concepts covered in the lectures and exercises. The test will include multiple-choice questions, open-ended questions, and applied exercises.
The final grade will be based on the integration of the test results with the quality of the report and the laboratory notebook.

Students with physical disabilities, Learning Disabilities or Special Education Needs can request
specific services and tools via the
, consulting the University webpage: https://www.uniupo.it/en/services/services-students-physical-or-learning-disabilities

Students with disabilities, learning disabilities or special education needs, once they have contacted the University Staff, can refer to the tutor in charge of the course to define the examination
modalities, concerning academic aspects.

For students meeting the mandatory attendance threshold (75%), the final evaluation integrates two components:
- Continuous assessment of the laboratory notebook and simulation reports: Evaluates the accuracy in documenting experimental work, direct protocol comprehension, and applied practical skills.
- Written test (administered on the final lab day): Includes multiple-choice questions to assess theoretical knowledge, practical exercises (e.g., calculation of dilutions or kinetic constants) to evaluate the capacity to apply knowledge, and open-ended questions designed to verify making judgements when interpreting experimental outputs and the effectiveness of scientific communication.
The final evaluation is derived from the weighted integration of the written test score with the quality of the submitted lab notebook and reports.

Getting to know the laboratory and equipment: measuring, diluting, and preparing a calibration curve. Using Excel for constructing a calibration curve and practical exercises.
How to keep a laboratory notebook.
How to read and interpret a protocol.
Theory and practical exercises on solution preparation.

The significance of measurement in a biological context: specificity, accuracy, precision, and reproducibility.

Techniques for sample preparation for biochemical analysis (homogenization, detergent lysis). Solvent extraction and selective precipitation, their application to the fractionation of biological compounds. Theory of centrifugation and preparative centrifugation techniques for the fractionation of biological compounds. Use of centrifuges and centrifugal force.

Principles of spectrophotometry. The theoretical foundations and practical aspects of spectroscopic techniques (absorption, fluorescence, polarimetry) and examples of their use for the quantification of biological analytes. Characteristics and use of a spectrophotometer and fluorimeter.

Principles of chromatography, main chromatographic techniques (affinity, gel filtration, ion exchange, and reverse phase) and related matrices. Use of chromatography for separating complex mixtures of proteins and lipids based on their chemical and physical properties. Equipment used (column chromatography, thin layer chromatography, HPLC, etc.). Detectors and results analysis (retention coefficients, qualitative and quantitative analysis).
Principles of electrophoresis with a focus on protein fractionation and characterization. IEF, SDS-PAGE, 2D-PAGE, capillary electrophoresis. Common detection systems: dyes and western blotting.

Applications of antibodies in biotechnology, competitive and non-competitive immunoassays, RIA, ELISA, immunofluorescence, Western blotting, immunodiffusion, and related techniques.

Use of enzymes in diagnostics and research—characterization of enzyme kinetics and study of inhibitors. Use of enzymes in clinical diagnostics, kinetic and end-point assays applied to research and diagnostic analysis of an enzyme's kinetic parameters.

Binding and association assays, analysis of receptor-ligand equilibrium.

By the end of the course, students are expected to achieve the following learning outcomes:
1. Knowledge and understanding: Describe the theoretical and physical principles of spectrophotometry, separation techniques (chromatography, centrifugation), and analytical methods (electrophoresis, Western Blotting, immunological and enzymatic assays). Explain the quality parameters of biological measurements (specificity, accuracy, precision, reproducibility) and the fundamentals of enzyme kinetics and binding assays. Identify the appropriate equipment for the preparation, lysis, and fractionation of complex biological samples.
2. Applying knowledge and understanding: Prepare biological solutions and perform dilutions by accurately calculating concentrations. Generate and interpret a spectrophotometric calibration curve using spreadsheet software (Excel) for analyte quantification. Apply experimental protocols for protein separation and characterization using electrophoretic (SDS-PAGE) and immunological (ELISA, Western Blot) techniques. Draft a clear, organized, and scientifically rigorous laboratory notebook and experimental report.
3. Making judgements: Critically evaluate the quality and reliability of biochemical data collected during practical lab sessions and software simulations. Select the most appropriate chromatographic, electrophoretic, or immunological method to separate or analyze macromolecular mixtures based on their physical-chemical properties. Interpret enzymatic kinetic parameters and inhibition plots to deduce the underlying mechanism of action.
4. Communication skills: Present experimental data and describe biochemical procedures using proper scientific terminology and analytical/mathematical formalisms. Clearly and efficiently communicate methodological information retrieved from experimental protocols or scientific literature.
5. Learning skills: Acquire the ability to independently understand, interpret, and implement new experimental protocols and analytical methods described in scientific literature or technical manuals. Develop a rigorous and adaptable working methodology suitable for future experimental thesis projects.