Transcript KAEDAH-KAEDAH DALAM BIOLOGI MOLEKUL

TECHNIQUES IN
MOLECULAR BIOLOGY
• CENTRIFUGATION- Separation of
molecules/macromolecules/organelles
according to the size, shape, density &
gradient
• ELECTROPHORESIS- Separation of
molecules/macromolecules according to
charge
• MICROSCOPY- Structural examination of
minute molecule/macromolecule/organelle
CENTRIFUGATION
• MATERIALS OR PARTICLES IN A SOLUTION
CAN BE SEPARATED BY A CENTRIFUGE
THAT USES THE PRINCIPLE OF
CENTRIFUGATION
• CLASSES:
-ANALYTICAL/PREPARATIVE
-ULTRACENTRIFUGATION AND LOW SPEED
-DIFFERENTIAL/ZONAL CENTRIFUGATION
http://ntri.tamuk.edu/centrifuge/centrifugation.htm
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ANALYTICAL CENTRIFUGATION
• IS USED TO MEASURE THE SEDIMENTED
PARTICLE PHYSICAL CHARACTERISTICS
SUCH AS SEDIMENTATION COEFFICIENT
AND MOLECULAR WEIGHT
PREPARATIVE
CENTRIFUGATION
• TO SEPARATE SPECIFIC PARTICLES
THAT IS REUSABLE
• TYPES:
- RATE ZONAL
- DIFFERENTIAL
- ISOPYCNIC CENTRIFUGATION
ULTRACENTRIFUGATION AND
LOW SPEED
• DEPENDS ON SPEED
• ULTRACENTRIFUGATION - THE SPEED
EXCEEDS 20,000 RPM
• SUPER SPEED ULTRACENTRIFUGATIONTHE SPEED IS BETWEEN 10,000 RPM20,000 RPM
• LOW SPEED CENTRIFUGATION- THE
SPEED IS BELOW 10,000 RPM
DIFFERENTIAL
CENTRIFUGATION
• PARTICLES IN SAMPLE WILL SEPARATE INTO
SUPERNATANT AND PELLET OR IN BOTH
DEPENDING ON THEIR SIZE, SHAPE,
DENSITY AND CENTRIFUGATION CONDITION
• THE PELLET CONTAINS ALL THE
SEDIMENTED COMPONENT MIXTURE AND
CAN CONTAIN MATERIALS THAT WAS NOT
SEDIMENTED EARLIER
DIFFERENTIAL
CENTRIFUGATION
• SUPERNATANT CONTAINS MATERIALS THAT
ARE NOT SEDIMENTED BUT CAN BE
SEDIMENTED WHEN CENTRIFUGATION IS
DONE AT A HIGHER SPEED
DIFFERENTIAL
CENTRIFUGATION
ZONAL CENTRIFUGATION
• SAMPLE IS APPLIED ON TOP OF SUCROSE
OR CESIUM CLORIDE SOLUTION
• PARTICLE CAN BE SEPARATED ACCORDING
TO SIZE & SHAPE (TIME-RATE ZONE) OR
DENSITY (ISOPYCNIC)
RATE-ZONAL
CENTRIFUGATION
ISOPYCNIC-ZONAL
CENTRIFUGATION
SEDIMENTATION COEFFICIENT
• WHEN CELL COMPONENTS ARE
CENTRIFUFED THROUGH A GRADIENT
SOLUTION, THEY WILL SEPARATE INTO
THEIR OWN ZONE OR LINE/LAYER
• THE RATE WHEN THE COMPONENT
SEPARATES IS CALLED AS SEDIMENTATION
COEFFICIENT OR THE s VALUE (SVEDBERG
UNIT )
1 S = 1 X 10-13 SECONDS
SEDIMENTATION COEFFICIENT
VALUES
PARTICLE OR
MOLECULE
SEDIMENTATION
COEFFICIENT
LYSOSOME
TOBACCO MOSAIC VIRUS
RIBOSOME
RIBOSOMAL RNA MOLECULE
tRNA MOLECULE
HEMOGLOBIN MOLECULE
9400S
198S
80S
28S
4S
4.5S
SPEED OF CENTRIFUGATION
• A PARTICLE THAT IS ROTATING WILL HAVE
A PULLING FORCE IN A FORM OF
MAGNITUDE TO SPEED FUNCTION AT
DEFINED ANGLE (ROTATION SPEED) AND
CENTRFUGATION RADIUS (THE DISTANCE
BETWEEN THE SAMPLE CONTAINER AND
THE ROTOR CENTRE)
SPEED OF CENTRIFUGATION
• 2 WAYS OF EXPRESSING THE PULLING
FORCE:
a) RELATIVE CENTRIFUGATIONAL FORCERCF (g)
b) ROTATION PER MINUTE (rpm)
RELATIVE
CENTRIFUGATIONAL FORCE
• THE PULLING FORCE OF
CENTRIFUGATION IS BASED ON OR
RELATIVE TO THE STANDARD
GRAVITATIONAL FORCE
• FOR EXAMPLE 500x g MEANS THAT THE
PULLING FORCE IS 500 TIMES BIGGER
THAN THE STANDARD GRAVITATIONAL
FORCE
RELATIVE
CENTRIFUGATIONAL FORCE
• EQUATION
R.C.F. = 1.119 x 10 -5 (rpm2) r
rpm=rotation per minute
r=radius (in cm)
UNIT g
ELECTROPHORESIS
• THE MOVEMENT OF CHARGED PARTICLE
IS INFLUENCED BY ELECTRICAL CURRENT
• ELECTROPHORESIS IS THE METHOD OF
SEPARATING MACROMOLECULE SUCH AS
NUCLEIC ACID AND PROTEIN ACCORDING
TO SIZE, ELECTRICAL CHARGE AND
PHYSICAL PROPERTIES SUCH AS DENSITY
ETC
• SEPARATION IS AIDED BY A MATRIX SUCH
AS POLIACRYLAMIDE OR AGAROSE
ELECTROPHORESIS
• PRINCIPLE: SEPARATION OF
MACROMOLECULE DEPENDING ON TWO
PROPERTIES: WEIGHT AND CHARGE
• ELECTRICAL CURRENT FROM THE
ELECTRODE WILL PUSH THE MOLECULE AND
AT THE SAME TIME THE OTHER ELECTRODE
WILL PUT IT
• MOLECULES WILL MOVE ALONG THE PORES
THAT ARE FORMED BETWEEN THE INTERWOVEN MATRIX THAT ACTS LIKE A SIEVE TO
SEAPARATE THE MOLECULE ACCORDING TO
THEIR SIZE
ELECTROPHORESIS
• ELECTRICAL CURRENT WILL FORCE THE
MACROMOLECULE TO MOVE ALONG THE
PORES
• THE MACROMOLECULE MOVEMENT DEPENDS
ON THE ELECTRICAL FIELD FORCE, THE
MOLECULE SIZE AND SHAPE, THE SAMPLE
RELATIVE HYDROPHOBIC PROPERTY, IONIC
STRENGTH AND THE TEMPERATURE OF THE
ELECTROPHORESIS BUFFER
• DYEING WILL AID THE VISUALISATION OF
MACROMOLECULE IN THE FORM OF
SEPARATED SERIES OF STRIPES
PROTEIN ELECTROPHORESIS
• PROTEIN HAS A POSITIVE OR NEGATIVE
NET CHARGE AS A RESULT OF THE
COMBINATION OF CHARGED AMINO ACIDS
CONTAINEDIN THEM
• THE MATRIX THAT IS USUALLY USED FOR
PROTEIN SEPARATION IS POLIACRYLAMIDE
• TWO DIMENSIONAL GEL
ELECTROPHORESIS- PROTEIN SEPARATION
ACCORDING TO ISOELECTRICAL POINTS
AND MOLECULAR WEIGHT
2-D PROTEIN
ELECTROPHORESIS
• FIRST STEP/DIMENSION:
PROTEIN SEPARATION ACCORDING TO
ISOELECTRIC POINT (PROTEIN CONTAINS
DIFFERENT POSITIVE AND NEGATIVE
CHARGE RATIO)
-ELECTROPHORESIS IS DONE ON THE GEL
IN THE FORM OF TUBE; PROTEIN WILL
MOVE IN A SOLUTION WITH DIFFERENT pH
GRADIENT
2-D PROTEIN
ELECTROPHORESIS
• FIRST STEP/DIMENSION:
-PROTEIN WILL STOP WHEN IT REACHES
THE pH WHICH IS EQUAL TO ITS
ISOELECTRIC POINT i.e WHEN THE
PROTEIN DOES NOT HAVE A NET CHARGE.
+
-
BASIC
ACIDIC
2-D PROTEIN
ELECTROPHORESIS
• SECOND STEP/DIMENSION:
• PROTEIN SEPARATION BY MOLECULAR
WEIGHT
• ELECTROPHORESIS IS DONE IN AN
ORTHOGONAL DIRECTION FROM
THE FIRST STEP;
SODIUM DODECYL SULPHATE
(SDS) IS ADDED
+
-
2-D PROTEIN
ELECTROPHORESIS
1-D PROTEIN
ELECTROPHORESIS
• PROTEIN IS SEPARATED BY ITS MOLECULAR
WEIGHT ONLY
• THE TECHNIQUE IS ALSO KNOWN AS
POLIACRYLAMIDE GEL ELECTROPHORESIS
(PAGE) OR SDS-PAGE IF SDS IS PRESENT
DURING SAMPLE PREPARATION
• SIMULATION OF 1-D ELECTROPHORESIS
http://www.rit.edu/~pac8612/electro/
Electro_Sim.html
SDS-PAGE
• TO SEPARATE PROTEIN WITH THE SIZE OF 5
- 2,000 kDa
• PORES IN BETWEEN THE POLIACRYLAMIDE
MATRIX CAN VARIES FROM 3%-30%
• THE PROTEIN SAMPLE IS IN THE FORM OF
PRIMARY STRUCTURE (SAMPLE IS BOILED
WITH SDS AND -MERCAPTOETHANOL
PRIOR BEING LOADED ONTO GEL)
SDS-PAGE
• PROTEIN IS STAINED USING COOMASIE
BLUE OR SILVER
• NON-DIRECTIONAL STAINING CAN BE
DONE:
-ANTIBODY BOUND WITH RADIOISOTOPE
OR ENZYME, FLUORESENCE DYE
SDS-PAGE
• SDS FUNCTION:
NEGATIVELY CHARGED
DETERGENT THAT
BINDS TO THE
HYDROPHOBIC REGION
OF THE PROTEIN
MOLECULE; AS A
RESULT THE PROTEIN
BECOMES A LONG
POLIPEPTIDE CHAIN AND FREE
FROM OTHER PROTEINS AND
SDS-PAGE
• -MERCAPTOETHANOL FUNCTION: TO
BREAK DISULPHIDE BONDS SO THAT
PROTEIN SUBUNIT CAN BE ANALYSED
NUCLEIC ACID
ELECTROPHORESIS
• AGAROSE OR POLIACRYLAMIDE IS THE
MATRIX USUALLY USED TO SEPARATE
NUCLEIC ACID IN A TECHNIQUE KNOWN AS
AGAROSE GEL ELECTROPHORESIS
• SAMPLE CONTAINING DNA IS LOADED INTO
WELLS LOCATED NEAR TO THE
NEGATIVELY CHARGED ELECTRODE
• DNA THAT IS NEGATIVELY CHARGED WILL
BE ATTRACTED TO THE POSITIVE
ELECTRODE
NUCLEIC ACID
ELECTROPHORESIS
• DNA WITH A BIGGER SIZE WILL MOVE
SLOWER THAN THE SMALLER SIZE WHICH
MOVE FASTER
• STAINING IS DONE USING ETHIDIUM
BROMIDE (EtBr) THAT ENABLES THE
VISUALISATION OF NUCLEIC ACID; EtBr IS
INSERTED BETWEEN THE BASES ON THE
NUCLEIC ACID
• EtBr IS ORANGE IN COLOUR WHEN LIT-UP
BY ULTRA-VIOLET LIGHT
NUCLEIC ACID
ELECTROPHORESIS
MICROSCOPY
• ONE OF THE EARLIEST TECHNIQUE TO
STUDY MACROMOLECULE
• PRINCIPLE: TO ENLARGE SMALL
IMAGES
• TYPES OF MICROSCOPY ACCORDING TO
THE SIZE OF IMAGE ENLARGEMENT
- LIGHT MICROSCOPE (300nm-2mm)
- ELECTRON MICROSCOPE
(0.15nm-100m)
LIGHT MICROSCOPE
• IMAGE ENLARGEMENT PRINCIPLE:
LIGHT FROM BELOW OF THE
MICROCOPE GOES THROUGH
THE CONDENSOR TO FOCUS THE
LIGHT TO THE SPECIMEN.
• LIGHT FROM THE SPECIMEN IS
RECOLLECTED BY THE OBJECTIVE
LENSE TO FORM AN IMAGE
LIGHT MICROSCOPE
• TYPES OF LIGHT MICROSCOPE :
BRIGHT-FIELD MICROSCOPE
DARK-FIELD MICROSCOPE
PHASE-CONTRAST MICROSCOPE
FLUORESENCE MICROSCOPE (UV)
(FLUORESCIN/RHODAMIN)
ELECTRON MICROSCOPE
• PRINCIPLE:
-ELECTRON IS USED (NOT LIGHT) TO
ENLARGE IMAGE
-SPECIMEN MUST UNDERGO A SERIES
OF PREPARATION PROCESSES SUCH AS
COATING WITH THIN LAYER OF GOLD TO
ALLOW EMITTED ELECTRON TO
COLLIDE TO AND THEN RECOLLECTED
TO FORM IMAGE ON THE SCREEN
ELECTRON MICROSCOPE
• TYPES:
1) TRANSMISSION ELECTRON MICROSCOPE
-ELECTRON GOES THROUGH THE SPECIMEN
AND IMAGE IS RECOLLECTED ON A
FLUORECENS SCREEN
-THE INNER STRUCTURE OF THE SPECIMEN
CAN BE SEEN
ELECTRON MICROSCOPE
• TYPES:
2) SCANNING ELECTRON MICROSCOPE
-ELECTRON IS FOCUSSED TO THE
SPECIMEN AND THEN REEMITTED
(SCANNED) TO THE DETECTOR AND
IMAGE IS SEND TO THE SCREEN FOR
VIEWING
-THE OUTER STRUCTURE CAN BE SEEN
ELECTRON MICROSCOPE
SCANNING ELECTRON
MICROSCOPE
MOSQUITO IMAGES
BY SCANNING ELECTRON
MICROSCOPE
OTHER TECHNIQUES
• CHROMATOGRAPHY
-PAPER: PROTEIN SEPARATION BY USING
FILTER PAPER AS THE MATRIX
-ION-EXCHANGE
-GEL FILTRATION
-AFFINITY
-HIGH PRESSURE LIQUID
CHROMATOGRAPHY (HPLC)
OTHER TECHNIQUES
• RADIOISOTOPES FOR MOLECULE TAGGING :
32P, 131I, 35S, 14C, 45Ca, 3H
- RIA, ‘PULSE-CHASE’ EXPERIMENT,
AUTORADIOGRAPHY
• ANTIBODY (MONOCLONE/POLYCLONE) FOR
TAGGING MOLECULE: EIA, IF, ELISA
• X-RAY DIFFRACTION ANALYSIS: PROTEIN
STRUCTURE DETERMINATION
• DNA RECOMBINANT TECNOLOGY