Biochemical Techniques

Teacher

Dr Sitaram Swain

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Core Courses

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Course Name : Biochemical Techniques

Code(Credit) : CUTM1697(3-2-1)

Course Objectives

  • Students will learn about the principle and applications of spectrophotometry, different chromatographic techniques like gel filtration, Ion exchange, thin layer, etc.
    Students will also learn about various electrophoretic techniques such as agarose, gel, PAGE, etc. and their applications in analyzing proteins and nucleic acids.
    Students will learn the basic principles of centrifugation, various types of centrifuges, rotors and methods for subcellular fractionation. Principle and application of ELISA in biological experiments.
    This course contains bio analytical techniques along with their theory, working principal, common instrumentation and possible applications. This course will be equally beneficial to various scientific areas.
    Students will be exposed to various biological techniques and their applications in identification, isolation of different biological molecules.

Learning Outcomes

  • The student will obtain hands-on training in basic separation techniques in biochemistry and gain expertise in the isolation of various biomolecules and organelles.
    Students will know the principle and application of various instruments and they can be able to make strategy molecular techniques for the improvement in any trait or its well being based on the techniques learned during this course. Students can use this knowledge for biological experiments.

Course Syllabus

Module-I Chromatography: General Principles of chromatography, adsorption and partition. Techniques: Paper chromatography, Ascending, descending and circular, 2D chromatography, Rf values, column chromatography.
Module-II Principle of gel filtration, ion-exchange chromatography, affinity chromatography.
Thin layer chromatography (TLC) – Principle, procedure and applications.
Module-III Electrophoresis, Paper and gel electrophoresis, SDS-PAGE, 2D electrophoresis.
Module-IV Centrifugation: Principle of differential centrifugation. Types of Rotors, Ultra centrifuge –construction and applications in sub-cellular fractionation.
Module-V Radiochemistry: Natural and artificial radioactivity, characteristics of radioactive elements, units of radioactivity, disintegration constant, half-life, α, β and γ radiation. Detection of radioactivity by GM counter. Applications of radioisotopes – 3H, 14C, 131I, 60Co and 32P. Biological effects of radiations. Safety measure in handling radio isotopes.
Module-VI Types of ELISA, principle and application of ELISA,
Module-VII Spectrometry–Beer and Lamberts law, Principals of colorimeter, spectrophotometer. Nephelometry and turbidimetry and its applications
PRACTICAL CONTENT:
1. Identification of amino acids by paper chromatography.
2. Ascending paper chromatography of amino acids.
3. Separation of plant pigments using paper chromatography.
4. Separation of plant pigments by column chromatography using silica gel-G.
5. Polyacrylamide gel electrophoresis of proteins.
6. SDS-PAGE
7. Demonstration of separation of lipids by TLC.
8. Demonstration of two dimensional chromatography of amino acids
Recommended books:
Text Books:
Practical Biochemistry – Principles and Techniques, Wilson and Walker, Cambridge, University Press, Cambridge
Reference Books:
Biophysical Chemistry, Principles & Techniques – Upadhyay, Upadhyay and Nath Himalaya Publ. House.
Practical Biochemistry – Principles and Techniques, Wilson and Walker, Cambridge University Press, Cambridge
Boyer, R.F., Biochemistry Laboratory: Modern Theory and Techniques, 6th Ed., Boston, Mass: Prentice Hall, 2012.
Plummer D. T., An Introduction to Practical Biochemistry 3rd ed., Tata McGraw
Hill Education Pvt. Ltd. (New Delhi), 1998.

Session Plan

Session 1

Chromatography: General Principles of chromatography

Principle of chromatography

Chromatography

Session 2

Adsorption and partition. Techniques: Paper chromatography

Partition chromatography

Adsorption chromatography

Adsorption chromatography-video

Session 3

Flipped class/Assignment:Adsorption and partition.Techniques: Paper chromatography

Session 4

Practice:Identification of amino acids by paper chromatography.

PRACTICE

Session 5

Ascending, descending and circular, 2D chromatography

Paper chromatography

Session 6

Ascending paper chromatography of amino acids.

PRACTICE

Session 7

Rf values, column chromatography

Column chromatography

Session 8

Separation of plant pigments using paper chromatography.

PRACTICE

Session 9

Principle of gel filtration chromatography

Gel filtration chromatography

Session 10

Flipped Class/Assignment:Application of Principle of gel filtration chromatography

Session 11

Session 12

Separation of plant pigments by column chromatography using silica gel-G.

PRACTICE

PRACTICE

Session 13

Ion-Exchange chromatography-animation

Principle, procedure and applications:Ion-exchange chromatography

Session 14

Principle, procedure and applications: Affinity chromatography

PRACTICE

Session 15

Thin layer chromatography (TLC)

Thin layer chromatography

TLC Video

Session 16

Separation of amino acids by Thin layer chromatography (TLC)

PRACTICE

Session 17

Session 18

Session 19

Paper and gel electrophoresis

Gel electrophoresis

paper electrophoresis

Session 20

Practice

PRACTICE

Session 21

Session 22

Polyacrylamide gel electrophoresis of proteins

PRACTICE

Session 23

2D electrophoresis and IFE

2D gel electrophoresis

2D electrophoresis

Session 24

Demonstration of two dimensional chromatography of amino acids

PRACTICE

Session 25

PRACTICE SDS-PAGE

SDS PAGE Demonstration

Session 26

Centrifugation

Centrifugation

Session 27

Principle of differential centrifugation

CENTRIFUGE PRINCIPLE

Session 28

PRACTICE

centrifuge

Session 29

Types of Rotors, Ultra centrifuge –construction and applications in sub-cellular fractionation

principles and applications of centrifugation

ultra centrifugation

Session 30

Flipped Class-Ultra centrifuge

ultra centrifugation

Session 31

Radiochemistry: Natural and artificial radioactivity

Radioactivity

Radioactivity-artificial & natural

Session 32

characteristics of radioactive elements, units of radioactivity, disintegration constant

Radioisotopes in biologicalsystem

Session 33

half-life, α, β and γ radiation

Radiation

Session 34

Detection of radioactivity by GM counter

Detection of radio activity

Session 35

Flipped class-Detection of radioactivity

Session 36

Applications of radioisotopes – 3H, 14C, 131I, 60Co and 32P

uses of radioisotopes

Session 37

Assignment/ Flipped class-Applications of radioisotopes – 3H, 14C, 131I, 60Co and 32P

applications of radioisotopes

Session 38

Biological effects of radiations. Safety measure in handling radio isotopes.

Biological effects of radiation

radiation safety

Session 39

Flipped class-Biological effects of radiations and Safety measure in handling radio isotopes.

Session 40

General principles of ELISA

ELISA principle

Session 41

Session 42

Practice

PRACTICE

Session 43

Spectrometry

Spectroscopy

Session 44

Beer and Lamberts law

Beer's and Lambert law

Session 45

PRACTICE

Practice

Session 46

UV-VIS Spectrometry

spectroscopy

Session 47

Principles of colorimeter

colorimeter

Session 47

Principles of colorimeter

colorimeter

Session 48

Flipped class/Assignment-Colorimetric analysis

colorimetry

Session 49

Nephelometry and turbidimetry and its applications

principle

Nephelometry and Turbidimetry

Session 50

Turbidimetry and its applications

turbidimetry

Session 51

Turbidimetry and its applications

principle

nephelometry and turbidimetry

Case Studies

Case Studies

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    Dr Sitaram Swain

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    Dr Sitaram Swain has received PhD degree from Utkal University in the year 2017. He is also trained and experienced from TATA Memorial Hospital, Mumbai, India. He has experienced with cell line culture, clinical biochemistry, immuno-fixation, immunoassay, cancer cytogenetic, serum protein electrophoresis and tumour marker analysis. He has qualified NET(ICAR-ASRB) in Animal biotechnology. He has […]