The subject "Basic Biochemical Methodology" is mainly a practical subject. In Basic Biochemical Methodology, students will acquire basic knowledge about the techniques commonly used in biochemistry laboratories. The knowledge acquired in this subject is of vital importance for the subsequent academic and professional development.

The use of scientific literature, experimental design, laboratory safety, and ethics will be covered during the first semester.

During the second semester, the teaching activities are focused on acquiring sufficient knowledge and skills for the proper performance of the laboratory practices that will take place throughout the degree program. The various teaching activities in this semester of "Basic Biochemical Methodology" are closely aligned with the subject "Instrumental Techniques" offered in the second year. In "Instrumental Techniques," students will delve deeper into the concepts previously learned in "Basic Biochemical Methodology," specifically regarding chromatography, electrophoresis, and protein purification.

The primary goal of this subject is to equip students with the essential training and knowledge required to facilitate their future job integration into research centers, the biotechnology industry, or higher education institutions.



To achieve this, the following competencies will be addressed:

-Acquire the necessary knowledge to carry out good practices in a laboratory.

-Acquire skills in basic instrumentation and the most commonly used experimental methods.

-Utilize scientific and technical literature and apply it to the acquired knowledge.

-Design and/or conduct experiments, knowing how to describe, quantify, analyze, and critically evaluate the results obtained, and drawi conclusions based on them.

Syllabus:



BLOCK I: Scientific Articles and Journals. Bibliographic Search.

Topic 1: Scientific Articles and Journals: Structure of a scientific manuscript. How to write a research article. How to publish a research article. Types of scientific journals. The impact factor. Journals in electronic format.

Topic 2: Bibliographic Search: Databases of scientific articles. The PubMed database. Structure of a PubMed record. The PubMed search engine. Search strategies. Science Citation Index.



BLOCK II: The Scientific Method in Biochemical Research. Good Laboratory Practices in Biochemistry.

Topic 3: The Scientific Method in Biochemical Research: Formulation of a hypothesis. Experimental design. Treatment of results. Drawing conclusions. Ethical considerations.

Topic 4: Good Laboratory Practices in Biochemistry: Identification of hazards (physical, chemical, biological, and radiological). General and personal safety measures. Safety regulations. Behavior in emergency situations.



BLOCK III: Levels of Experimentation in Biochemistry. Cellular Systems. Subcellular Fractionation.

Topic 5: Levels of Experimentation in Biochemistry: Studies with intact animals, organs, isolated tissues, or cells. Molecular studies (structural or functional). Cellular systems. Techniques for separating different cell types (centrifugation, centrifugal elutriation, flow cytometry). Cell cultures. Cell quantification and viability. Cell lines. Quantification and viability of cells. The hemocytometer.



BLOCK IV: Basic Technology in the Biochemistry Laboratory:

Topic 6: Subcellular Fractionation: Methods for homogenization and obtaining crude extracts. Preparative centrifugation (differential and density gradient). Marker enzymes to identify various cellular organelles. Organelle viability. Analytical centrifugation.

Topic 7: Chromatography. Basic concepts and general principles. Chromatographic techniques based on polarity (liquid-solid chromatography, gas-liquid chromatography, liquid-liquid chromatography). Chromatographic techniques based on charge (ion-exchange chromatography). Chromatographic techniques based on size (molecular sieve chromatography). Affinity chromatography.

Topic 8: Electrophoretic Techniques. Basic concepts and general principles. Electrophoretic supports. Staining methods. Gradient electrophoresis. Polyacrylamide gel electrophoresis in the presence of SDS. Isoelectric focusing. Two-dimensional electrophoresis. Capillary electrophoresis.

Topic 9: Spectrophotometric Techniques. Basic concepts and general principles. Chromophores in biochemistry. Molar absorption coefficient. Lambert-Beer's law. Instrumentation: visible and ultraviolet spectroscopy.

Topic 10: Design of an Enzymatic Assay. Methods for the Quantification of Catalytic Activity. Experimental Determination of Kinetic Parameters.

Topic 11: Radiochemical Techniques. Atoms and Isotopes. Radioactive Decay. Types of Radioactive Emissions. Units of Radioactivity. Detection and Measurement of Radioactivity.

Topic 12: Immunochemical Techniques. Antibodies. Antigen-Antibody Interaction. Polyclonal and Monoclonal Antibodies. Antibody Labeling. Immunoprecipitation. Immunoassays (RIA, IRMA, ELISA).

Topic 13: Hybridization Techniques. Denaturation and Renaturation of DNA. Hypochromic and Hyperchromic Effects. Preparation and Labeling of Probes. Nucleic Acid Hybridization. Polymerase Chain Reaction (PCR). RT-PCR. DNA Chips.

Topic 14: Identification Techniques (Blotting). Basic Concepts. Blotting Transfer. Southern Blot. Northern Blot. Western Blot. Dot Blot.



Classroom Practical Session (GA):

Problem-solving exercises will allow for a deeper understanding of the concepts covered in the lectures:

-Centrifugation Problems

-Radioactivity Problems

-Spectrophotometry Problems

-Coupled Enzymatic Reactions Problems

-Primers Designing for PCR

Computer Practical Session (GO):

Scientific Articles and Journals. Bibliographic Searches in PubMed and ISI-WOK.

Laboratory Sessions (GL):

In the laboratory sessions, students will carry out the following seven practical activities:

Cell Disruption Methods. Protein Quantification.

Purification of Chloroplasts using Sucrose Gradient Centrifugation.

Isolation and Preparation of Mitochondria. Determination of Mitochondrial Viability.

Molecular Sieve Chromatography. Determination of Protein Molecular Weight.

Purification of Egg Lysozyme using Ion Exchange Chromatography.

Protein Electrophoresis on SDS-Polyacrylamide Gel.

Nucleic Acid Electrophoresis on Agarose Gel. Characterization of Plasmid DNA.

Seminars (S):

Students will develop their presentation skills through the exploration of topics directly related to the subject.

Topics 1 to 14 outlined in the syllabus will be explained in detail during lectures (M).

In classroom practices (GA), students will solve qualitative and quantitative exercises and problems related to the concepts covered in the lectures.

In seminars (S), students will present in groups a topic related to the subject.

In the laboratory sessions, students will carry out the seven practical activities described above. Attendance to the practical sessions is mandatory.

In computer practical session, students will use PubMed and ISI-WOK for biographical searches. Attendance to the computer practical sessions is mandatory.

• Continuous Assessment System
• Final Assessment System
• Tools and qualification percentages:
  • Exhibition of works, readings ... (%): 10
  • In this section, the sum of the percentages corresponding to theory (45%), laboratory practices (30%), computer exercises (5%), and problem-solving exercises (10%) is included. (%): 90

The assessment of the "Basic Biochemical Methodology" course is divided into five sections:



-Written Exam 45%: Correct answers, expression, argumentation, and use of scientific terminology will be taken into consideration. A minimum of 50% correct answers is required.

-Laboratory Practical Session 30%: Attendance, attitude, and cleanliness; accuracy and clarity of the report/exam will be taken into account, with a requirement to pass at least 50% of the questions.

-Computer Practical Session 5%: Attendance, attitude, and completion of exercises.

-Seminar 10%: Attendance, attitude, organization of information, analytical and synthesis skills, clarity of presentation, and participation in the debate.

-Problem-solving 10%: Attendance, correct resolution of problems posed during classroom exercises; exam, requiring a minimum of 50% correct answers.

In the case of taking partial exams, it will be necessary to obtain a minimum of 50% correct answers to pass the subject.

Attendance to the different teaching modalities throughout the course will be mandatory to be eligible to take the exams in the regular session. A minimum of 50% correct answers will be required to pass the exam.

Waiving: Failing to take the final exam is sufficient to waive the final grade.

Not taking the final exam is enough to receive a grade of "not presented" for the course.

The use of books, notes, phones, electronic devices, computers, or other equipment is not allowed during assessment tests (except for a calculator*). If any academic dishonesty or fraudulent practices are detected, the protocol on academic ethics and prevention of such practices at UPV/EHU will be enforced.

The final grade will be calculated by adding the grades obtained in the following sections:

-Written Exam 45%

-Laboratory Practical Session 30%.

-Computer Practical Session 5%.

-Seminar 10%.

-Problem-solving 10%.

Participation in the different teaching modalities throughout the course will be mandatory to be eligible to take the exams in the extraordinary session. A minimum of 50% correct answers will be required to pass the exam.

The grades of the sections passed will be kept for the extraordinary assessment session of that school year if the subject is failed in the ordinary call.

Not taking the written test will be qualified as "not presented" in the final grade for the course.

The eGela webpage (http://egela.ehu.eus) will be used to publish the course guide and information about the activities performed in the laboratory, computer room and classroom.
Before entering the laboratory, students must carefully read the protocol for the corresponding session. This protocol will be uploaded to eGela.

Basic bibliography

- Wilson, K. and Walker, J. (eds.)(2010). Principles and Techniques of Biochemistry and Molecular Biology. 7th edn. Cambridge University Press

- Roca, P. y cols. (2003). Bioquímica. Técnicas y Métodos. Editorial Hélice

- García-Segura, J.M. y cols. (2002). Técnicas instrumentales de análisis en Bioquímica. Editorial Síntesis

- Freifelder, D. (2003). Técnicas de Bioquímica y Biología Molecular. Editorial Reverté.

In-depth bibliography

- Boyer, R. F. (2009). Biochemistry laboratory: modern theories and techniques. Pearson Education.

Journals

The journal of biological chemistry, Crc critical reviews in biochemistry, European journal of biochemistry/febs, Journal of biochemical and biophysical methods, Bioscience, biotechnology, and biochemistry, Progress in biochemical pharmacology, Archives of biochemistry and biophysics, European journal of medicinal chemistry, Clinical physiology and biochemistry, J capillary electrophor, , Appl theor electrophor, J. of chromatography, Analytica chimica acta.

Web addresses

http://ocw.mit.edu/OcwWeb/web/home/home/index.htm
http://www.sciencedirect.com/science/journal/00219673
http://workbench.concord.org/database/
http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-376-9_6
http://www.ncbi.nlm.nih.gov/pubmed
https://apps.webofknowledge.com/