Introduction to Biochemical techniques

Transcription

1 Introduction to Biochemical techniques By Assist Prof. Dr. Khomsorn Lomthaisong Division of biochemical researches Protein and peptides research Molecular and Nucleic acid research 1

2 Electrophoresis set Electrophoresis kit 2

3 Protein and peptide research techniques - Chromatography Column chromatography Gel filtration Ion exchange chromatography HPLC GC Thin layer chromatography - Detection of protein - Electrophoresis of protein - Enzyme linked immunosorbent assay - Western blot - Immunocytochemistry 3

4 Nucleic acid research Nucleic purification Molecular cloning DNA sequencing Polymerase chain reaction Reverse transcription polymerase chain reaction Differential display RT-PCR Real time polymerase chain reaction Detection of nucleotides Southern blotting In situ hybridisation Chromatography 4

5 Principles of Chromatography Need: 1. Mobile phase (MP) solvent carrying a sample through the column. Either a gas or liquid 2. Stationary phase (SP) substance which stays fixed in column and creates the separation. Solid or viscous liquid -coated on a solid support. Partitioning between solid and liquid creates separation. "Like is attracted to Like" Property of chromatography matrix Rigidity Low non-specific intereaction Chemical stability Open pore structure 5

6 Most used packing materials Cellulose Cross-linked dextrans (sephadex) Agarose (Sepharose) Polyacrylamide Chromatographic principles 6

7 Chromatographic principles Principles of chromatography 7

8 Chromatographic principles Chromatographic principles 8

9 Chromatographic principles Describing a chromatogram Chromatogram -detector response vs. time (or volume) each peak represents a different substance eluted. retention time tr is amount of time needed after injection for an individual solute to reach the detector 9

10 Resolution better the resolution the more complete the separation of nearby peaks. 10

11 Peak shape 11

12 The General elution problems 12

13 Gel filtration (Molecular exclusive chromatography) Principles of gel filtration 13

14 Principles of gel filtration Gel filtration process Pack the the gel particles into the column Apply samples to the column Buffer (mobile phase) move through the column carry out large molecules and leave the smaller molecules to stage longer 14

15 Affinity chromatographic 15

16 Affinity chromatographic terminology Elution: buffer conditions are changed to reverse (weaken) the interaction between the target molecules and the ligand so that the target molecules can be eluted from the column. Wash: buffer conditions that wash unbound substances from the column without eluting the target molecules or that re-equilibrate the column back to the starting conditions (in most cases the binding buffer is used as a wash buffer). Ligand coupling: covalent attachment of a ligand to a suitable preactivated matrix to create an affinity medium. Pre-activated matrices: matrices which have been chemically modified to facilitate the coupling of specific types of ligand. 16

17 Applications of Affinity chromatography Enzyme substrate analogue, inhibitor, cofactor. Antibody antigen. Lectin polysaccharide, glycoprotein, cell surface receptor, cell. Nucleic acid complementary base sequence, histones, nucleic acid polymerase, nucleic acid binding protein. Hormone, vitamin receptor, carrier protein. Glutathione glutathione-s-transferase or GST fusion proteins. Metal ions Poly (His) fusion proteins, native proteins with histidine, cysteine and/or tryptophan residues on their surfaces. Reverse phase chromatography depends on the hydrophobic binding interaction between the solute molecule in the mobile phase and the immobilised hydrophobic ligand, i.e. the stationary phase 17

18 Interaction of a solute with a typical reversed phase medium. Principle of reversed phase chromatography with gradient elution. 18

19 Ligands used in reverse phase chromatography Typical n-alkyl hydrocarbon ligands. (A) Two-carbon capping group, (B) (B) Octyl ligand, (C) Octadecyl ligand. Ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge. 19

20 Ion exchanger types The principle of ion exchange chromatography 20

21 Protein Purification require more than one step each step in the process will cause some loss of product. e.g. if a yield of 80% ineachstepisassumed, this will be reduced to only 20% to reach the targets for yield and purity, the minimum number of steps and the simplest possible design is essential, until purity requirements have been fulfilled. 21

22 Preparation and the Three Phase Purification Strategy Three Phase Purification Strategy capture phase to isolate, concentrate and stabilise the target product. intermediate purification phase to remove most of the bulk impurities such as other proteins and nucleic acids, endotoxins and viruses. polishing phase to achieve high purity by removing any remaining trace impurities or closely related substances. 22

23 Guidelines for Protein Purification 1 Define objectives for purity, activity and quantity required of final product to avoid over or under developing a method 2 Define properties of target protein and critical impurities to simplify technique selection and optimisation 3 Develop analytical assays for fast detection of protein activity/recovery and critical contaminants 4 Minimise sample handling at every stage to avoid lengthy procedures which risk losing activity/reducing recovery 5 Minimise use of additives additives may need to be removed in an extra purification step or may interfere with activity assays 6 Remove damaging contaminants early for example, proteases 7 Use a different technique at each step to take advantage of sample characteristics which can be used for separation (size, charge, hydrophobicity, ligand specificity) 8 Minimise number of steps extra steps reduce yield and increase time, combine steps logically KEEP IT SIMPLE! 23

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25 Suitability of purification techniques for the Three Phase purification strategy Electrophoresis : Gel electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation 25

26 Proteins always composed with positive (anode) or negative (cathode) Nucleic acids have a consistent negative charge imparted by their phosphate backbone Proteins and nucleic acids are electrophoresed within a matrix or "gel" 26

27 Gel types Agarose: Polyacrylamide: - is a polysaccharide extracted from seaweed - have a large range of separation - DNA from about 200 to 50,000 bp can be separated is a cross-linked polymer of acrylamide typically used between 3.5 and 20% Have higher resolution Acrylamide is a potent neurotoxin Agarose Gel Electrophoresis of DNA DNA will migrate towards the anode, which is usually colored red 27

28 Migration of DNA Fragments in Agarose Agarose Concentration Voltage Electrophoresis Buffer Effects of Ethidium Bromide Effect of agarose concentration on migration of the DNA 28

29 Polyacrylamide Gel Elcetrophoresis (PAGE) Non-denaturing PAGE Denaturing PAGE Containing of sodium dedocy sulfate (SDS) or Urea to denature protein during electrophorased 29

30 Principles of protein separation Protein Separation depends on Charge on the Proteins Shape of the Proteins Size of the Proteins Electric Field Charge on the Proteins - At the pi of a specific protein, the protein molecule carries no net charge and does not migrate in an electric field. - At ph above the pi, the protein has a net negative charge and migrates towards the anode. - At ph below the ph, the protein obtains a net positive charge on its surface and migrates towards the cathode. 30

31 Shape of the Proteins proteins can also be separated based on their difference in shape. A long, loose protein tends to interact more with the gel network and travels at a slower rate than a globular protein. Size of the Proteins separation of proteins by size SDS-PAGE enables the separation of proteins by size 31

32 Docium dodecyl sulfate (SDS) is used as a detergent in electrophoresis to dissociate a protein Electric Field The rate of separation (v) quickly depends on the electrophoretic mobility (U) and electric field strength (E): v = U * E How can we improve the efficiency of electrophoresis? increase U and E 32

33 Polymerase chain reaction (PCR) What is DNA? 33

34 Structure of DNA 34

35 Principle of the PCR to make a huge number of copies of DNA Composed of three major steps i.e. Denaturation at 94 C Annealing at 54 C extension at 72 C, done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time The cycling reactions the double strand melts open to single stranded DNA 35

36 The cycling reactions Forwards and reverse primers bind to single stranded DNA templates The cycling reactions dntps (that complimentary to the template) are coupled to the 3 end of primers and extend as a new stranded DNA 36

37 PCR animation Amplification of PCR 37

38 Amplification of PCR there is an exponential increase of the number of copies of the DNA DNA copy = 2 (n+1) n=cycle number Verification of the PCR product Run PCR product on the agrose gel Estimate the product size comparing to standard DNA 38

39 Types of PCR Reverse transcription polymerase chain reaction (RT-PCR) Differential display RT-PCR Realtime PCR In situ PCR RACE (repid amplification cdna end) RAPD (random amplification polymorphism DNA) AFLP (aplification fragment length polymerphism) Application of PCR Examination of particular diseases Amplify gene and DNA Cloning Analysis of phylogenetic relationships DNA fingerprinting Analysis of gene expression pattern and so on??????? 39

40 Application of PCR in Research Project การใช เทคน ค Differential display RT-PCR เพ อศ กษาการ เปล ยนแปลงการแสดงออกของย น Total rrna extraction 28s rrna 18s rrna ร ปท 1 ผลการตรวจสอบความบร ส ทธ ของ total RNA บน ด วย ว ธ agarose gel electrophoresis Lane 1 = total RNA จากกล มควบค ม Lane 2 = total RNA จากกล มท ได ร บโมโน โซเด ยมกล ตาเมต 40

41 100 bp ladder bp ladder 1500 bp 1500 bp 500 bp 500 bp 400 bp 400 bp DDRTPCR products gel 300 bp 300 bp 200 bp 200 bp 41