In this subject, the theoretical foundation of two typical tools used by biochemists is studied: chromatography and electrophoresis, as well as their practical applications. As a representative example of the preparative and analytical utility of these techniques, emphasis is placed on the purification and characterization of proteins and enzymes. The course also covers how to conduct laboratory assays to determine enzyme activity and contextualize it within enzymatic purification. A practical and quantitative approach is achieved through laboratory practices, problem-solving, and computer simulation.



This subject requires students to have knowledge not only in Biochemistry but also in Chemistry, Mathematics, and Physics. It is fundamental in the education of scientific professionals, as it explains preparative and analytical techniques essential for their career. The subject builds upon knowledge acquired in other previously taken courses (Biochemistry I, Biochemistry II, Basic Biochemical Methodology), complements concurrent subjects (Molecular Biology and Genetic Engineering, Recombinant DNA Technology), and lays the groundwork for subsequent subjects (Biocatalysis, Biotechnological Processes and Products, among others).

Cross-cutting skills:



-The student distinguishes and describes the principles of chromatographic and electrophoretic separation of proteins and explains their biochemical applications.

-The student correctly executes laboratory protocols related to the field of Biochemistry and Molecular Biology, applying them particularly to protein purification, considering criteria of purity and yield.

-The student handles, interprets, and explains the information extracted from scientific literature regarding protein separation, purification, and characterization techniques.



Specific skills:

-The student applies the results of an inquiry or research process in academic work, considering Sustainable Development Goals.

-The student synthesizes and communicates ideas orally and in writing, using academic language and integrating the terms covered in the subject.

Syllabus:



1. Protein purification and enzymatic assays.

2. Chromatographic techniques.

3. Electrophoretic techniques.





1.Protein purification and enzymatic assays

Strategies and steps for protein purification. Purification controls. Purity criteria. Purification tables. Techniques for isolation and purification of recombinant proteins.

Enzymatic activity with enzymatic extracts and with purified enzymes. Interfering and auxiliary enzymes. Coupled assays. Continuous and discontinuous methods. Reaction progress curves. Kinetic characterization of an enzyme.



2.Chromatographic techniques

Introduction. Definitions. History of chromatography. Theory of Chromatography. Classification of chromatographic techniques. Partition and adsorption chromatography. Chromatographic band and peak. Chromatographic equilibrium. Properties of the Gaussian curve. Chromatographic parameters: retention time and volume, retention factors, delay, and separation. Components of a chromatographic system. Adsorption chromatography. Hydroxyapatite chromatography. Hydrophobic interaction chromatography. Ion exchange chromatography. Affinity chromatography. Partition chromatography. Size exclusion chromatography. Paper and thin-layer chromatography. HPLC, UPLC, and FPLC. Gas chromatography. Supercritical fluid chromatography.



3.Electrophoretic techniques

Introduction. Theory of electrophoresis. Classification. Gel Electrophoresis. Two-dimensional electrophoresis. Electrophoresis in other media. Immunoelectrophoresis. Activity assay in gels. Capillary electrophoresis.

To follow the theoretical lectures, students have access to all slides, supplementary readings, and other teaching materials on the virtual classroom platform (e-Gela). In addition to theoretical lectures, students will engage in laboratory practices and computer simulations of protein purification. Students will receive information on gathering scientific articles related to protein purification for the Seminar assignment. Finally, students will make an oral presentation to summarize the scientific papers they have previously worked on.

• Continuous Assessment System
• Final Assessment System
• Tools and qualification percentages:
  • Final Exam (Written Exam): 60-65% Assignments (practical sessions, seminars...): 35-40% The total sum of the percentages will be (%): 100

Evaluation system



The lecture-based teaching will be evaluated through a final exam comprising questions related to lectures, practical sessions, and seminars, representing 60-65% of the final grade. The remaining percentage (35-40%) will be allocated to other assignments (seminars, laboratory practices, problem-solving, and computer simulation) to complete the 100%.



Passing the final exam with a score equal to or higher than 5 out of 10 is required to calculate the average with the other sections of the course.



A joint activity will be conducted with the subject Recombinant DNA Technology / Molecular Biology and Genetic Engineering to integrate the knowledge acquired in protein overexpression and purification. Additionally, the cross-cutting competency of Social Commitment, Communication, and Multilingualism will be addressed.



Students have the right to be evaluated through a end-of-course evaluation system. To do so, the student must submit in writing to the responsible faculty member of the course a waiver for continuous evaluation system. Students have a period of 9 weeks from the beginning of the semester to do so.



In case of waiving continuous evaluation system, the student will be evaluated through a final exam held on the same date as the continuous evaluation final exam. This exam will consist of the same exam as in the continuous evaluation system plus other exams and/or activities equivalent to the assignments evaluated in continuous evaluation system.



Attendance to laboratory practices is mandatory. The course cannot be passed if laboratory practices are not completed, regardless of the chosen evaluation system (continuous or end-of-course).



In any type of evaluation, not taking the final exam is enough to receive a grade of "not presented" for the course.



Academic Ethics and Dishonest Practices

This subject adheres to the "Protocol on Academic Ethics and Prevention of Dishonest or Fraudulent Practices in Assessment Tests and Academic Work at UPV/EHU," with the consequences outlined in section 5 of this protocol.

The grade obtained in the passed sections of continuous evaluation system (practical sessions and seminars) will be retained in the extraordinary assessment session of the same academic year.

There is no single book that can be classified as a reference textbook for this subject. An online platform (e-Gela) is available at the beginning of the course, which includes multimedia teaching materials, supplementary readings, and other didactic tools to follow the course. Commercially available software programs will be used for computer simulation.

Basic bibliography

PRINCIPLES AND TECHNIQUES OF BIOCHEMISTRY AND MOLECULAR BIOLOGY. Keith Wilson and John Walker. 7th Ed. Cambridge Univ. Press, 2010



BIOCHEMICAL TECHNIQUES: THEORY AND PRACTICE, Robyt, J.F. and White, B.J. Waveland Press, Prospect Heights, 1990



BIOCHEMISTRY LABORATORY: MODERN THEORY AND TECHNIQUES. Rodney F. Boyer. Benjamin Cummings, 2006

FUNDAMENTAL LABORATORY APPROACHES FOR BIOCHEMISTRY AND BIOTECHNOLOGY, A. J. Ninfa and D. P.Ballou, Wiley, 1998



PRINCIPLES AND TECHNIQUES OF PRACTICAL BIOCHEMISTRY, B.L. Williams and K. Wilson, Cambridge,Cambridge University Press, 2005



THE TOOLS OF BIOCHEMISTRY, Cooper, T. Wiley and Sons, New York, 1977



ENZYME ASSAYS. A PRACTICAL APPROACH. Eisenthal, R. and Danson, M.J. (eds) IRL Press, Oxford, 1992

In-depth bibliography

BIOTECHNOLOGY. A LABORATORY COURSE, Becker, J.M., Caldwell, G.A. & Zacho, E.A. Academic Press, San Diego,1996

PROTEIN METHODS, Bollag, D.M. and Edelstein, S.J. Wiley-Liss, Inc., New York, 1991

GUIDE TO PROTEIN PURIFICATION, Deutscher, M.P. (Ed.) Methods Enzymology, vol 182, Academic Press, London,1990

PROTEIN PURIFICATION METHODS. A PRACTICAL APPROACH, Harris, E.L.V.& Angal, S. (Eds) IRL Press, Oxford,1989

PROTEIN PURIFICATION APPLICATIONS. A PRACTICAL APPROACH, Harris, E.L.V. IRL Press, Oxford, 1990

PROTEIN PURIFICATION PROTOCOLS, Doonan, S. (Ed.). Humana Press Inc., Totowa, NJ, 1996

ENZYME PURIFICATION AND RELATED TECHNIQUES, Jakoby, W.B. Academic Press, London, 1989

PROTEIN PURIFICATION: PRINCIPLES, HIGH RESOLUTION METHODS AND APPLICATIONS, Janson, J.C. and Rydén, L. (Eds) Wiley VCH Publishers, Inc., New York, 1998

PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, Scopes, R. Springer-Verlag, New York, 1998

A PRACTICAL GUIDE TO MEMBRANE PROTEIN PURIFICATION, von Jagow, G. and Schägger, H. (Eds.) Academic Press, New York, 1994

Journals

Biochimica et Biophysica Acta, Journal of Biological Chemistry, Protein Expression and purification, Electrophoresis, Analytical Biochemistry, etc.

Web addresses

Simulation software for protein purification (Prácticas de ordenador)
http://agbooth.com/pp_java/

Cytiva Chromatography Handbooks
https://www.cytivalifesciences.com/en/us/support/handbooks

Nomenclature for chromatography
http://publications.iupac.org/pac/1993/pdf/6504x0819.pdf

Chromatography simulator – Chromulator
https://people.ohio.edu/gu/CHROM/index_chrom.html

HPLC simulator
https://hplcsimulator.org/