Introduction to

Biochemical techniques

By
Assist Prof. Dr. Khomsorn Lomthaisong

Division of biochemical
researches

z Protein and peptides research

z Molecular and Nucleic acid research

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Electrophoresis set

Electrophoresis kit

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 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

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

Chromatography

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z 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

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Most used packing materials

• Cellulose
• Cross-linked dextrans (sephadex)
• Agarose (Sepharose)
• Polyacrylamide

Chromatographic principles

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Chromatographic principles

Principles of chromatography

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Chromatographic principles

Chromatographic principles

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Chromatographic principles

Describing a chromatogram

z Chromatogram -detector response vs. time (or volume)

each peak
z represents a different substance eluted.
z retention time tr is amount of time needed after
injection for
z an individual solute to reach the detector

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Resolution
‘better the resolution the more complete the separation of
nearby peaks.’

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Peak shape

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The General elution problems

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Gel filtration (Molecular exclusive chromatography)

z Principles of gel filtration

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Principles of gel filtration

Gel filtration process

z Pack the the gel particles into the column

z Apply samples to the column

z Buffer (mobile phase) move through the column carry out

large molecules and leave the smaller molecules to
stage longer

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Affinity chromatographic

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 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 pre-

activated matrix to create an affinity medium.

Pre-activated matrices: matrices which have been chemically

modified to facilitate the coupling of specific types of ligand.

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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

z depends on the hydrophobic binding interaction between

the solute molecule in the mobile phase and the

immobilised hydrophobic ligand, i.e. the stationary phase

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Interaction of a solute with a typical reversed phase
medium.

Principle of reversed phase chromatography with

gradient elution.

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 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

z depends upon the reversible adsorption of charged solute

molecules to immobilized ion exchange groups of opposite

charge.

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Ion exchanger types

The principle of ion exchange

chromatography

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 Protein Purification
z require more than one step
z each step in the process will cause some loss of product.

e.g. if a yield of 80% in each step is assumed, this will be

reduced to only 20%

z to reach the targets for yield and purity, the minimum number
of steps and the simplest possible design is essential, until
purity
z requirements have been fulfilled.

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 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.

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 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!

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24
Suitability of purification techniques for the Three Phase
purification strategy

Gel electrophoresis
z Electrophoresis :
is a technique used to separate and sometimes
purify macromolecules - especially proteins and
nucleic acids - that differ in size, charge or
conformation

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z Proteins always
composed with positive
(anode) or negative
(cathode)

z Nucleic acids have a

consistent negative
charge imparted by their
phosphate backbone

Proteins and nucleic acids are

electrophoresed within a matrix or

"gel"

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 Gel types

z Agarose: z Polyacrylamide:

- is a polysaccharide extracted from

z is a cross-linked polymer of
seaweed
acrylamide

- have a large range of separation

z typically used between 3.5 and 20%

- DNA from about 200 to 50,000 bp can z Have higher resolution

be separated

z Acrylamide is a potent neurotoxin

Agarose Gel Electrophoresis of DNA

DNA will migrate towards the anode, which is usually colored red

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 Migration of DNA Fragments in
Agarose

z Agarose Concentration

z Voltage

z Electrophoresis Buffer
z Effects of Ethidium
Bromide

Effect of agarose concentration on

migration of the DNA

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Polyacrylamide Gel Elcetrophoresis (PAGE)
z Non-denaturing PAGE

z Denaturing PAGE
Containing of sodium dedocy sulfate (SDS) or Urea
to denature protein during electrophorased

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 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

z - 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.

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 Shape of the Proteins

z proteins can also be separated based on their difference in

shape.

z 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

z separation of proteins by size

z SDS-PAGE enables the separation of proteins by size

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Docium dodecyl sulfate (SDS) is used as a detergent in electrophoresis to
dissociate a protein

Electric Field

z The rate of separation (v) quickly depends on the

electrophoretic mobility (U) and electric field
strength (E):
v=U*E
z How can we improve the efficiency of
electrophoresis?
z increase U and E

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 Polymerase chain
reaction (PCR)

What is DNA?

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Structure of DNA

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Principle of the PCR
z to make a huge number of copies of DNA

z Composed of three major steps i.e.

• Denaturation at 94°C
• Annealing at 54°C
• extension at 72°C ,

z 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

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 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

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 PCR animation

Amplification of PCR

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 Amplification of PCR

z there is an exponential increase of the

number of copies of the DNA

n=cycle number
z DNA copy = 2 (n+1)

Verification of the PCR product

z Run PCR product on the agrose gel
z Estimate the product size comparing to standard DNA

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Types of PCR
z Reverse transcription polymerase chain
reaction (RT-PCR)
z Differential display RT-PCR
z Realtime PCR
z In situ PCR
z RACE (repid amplification cDNA end)
z RAPD (random amplification polymorphism
DNA)
z AFLP (aplification fragment length
polymerphism)

Application of PCR
z Examination of particular diseases
z Amplify gene and DNA
z Cloning
z Analysis of phylogenetic relationships
z DNA fingerprinting
z Analysis of gene expression pattern
z and so on???????

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 Application of PCR in Research
Project

การใชเทคนิค Differential display RT-PCR เพื่อศีกษาการ

เปลี่ยนแปลงการแสดงออกของยีน

Total rRNA extraction

28s rRNA

18s rRNA

รูปที่ 1 ผลการตรวจสอบความบริสุทธิ์ของ total RNA บน ดวย

วิธี agarose gel electrophoresis
Lane 1 = total RNA จากกลุมควบคุม
Lane 2 = total RNA จากกลุมที่ไดรับโมโน
โซเดียมกลูตาเมต

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 100 bp 100 bp
ladder ladder
1 2 3 4 5 6 7 8
1500 bp 1500 bp

500 bp 500 bp
DDRTPCR
400 bp 400 bp
products gel
300 bp 300 bp

200 bp 200 bp

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