techniques in molecular biology centrifugation- separation of molecules/macromolecules/organelles...
TRANSCRIPT
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TECHNIQUES IN TECHNIQUES IN MOLECULAR BIOLOGYMOLECULAR 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
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CENTRIFUGATIONCENTRIFUGATION
• 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.html
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ANALYTICAL CENTRIFUGATIONANALYTICAL CENTRIFUGATION
• IS USED TO MEASURE THE SEDIMENTED PARTICLE PHYSICAL CHARACTERISTICS SUCH AS SEDIMENTATION COEFFICIENT AND MOLECULAR WEIGHT
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PREPARATIVE PREPARATIVE CENTRIFUGATIONCENTRIFUGATION
• TO SEPARATE SPECIFIC PARTICLES THAT IS REUSABLE
• TYPES:
- RATE ZONAL
- DIFFERENTIAL
- ISOPYCNIC CENTRIFUGATION
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ULTRACENTRIFUGATION AND ULTRACENTRIFUGATION AND LOW SPEEDLOW SPEED
• DEPENDS ON SPEED• ULTRACENTRIFUGATION - THE SPEED
EXCEEDS 20,000 RPM• SUPER SPEED ULTRACENTRIFUGATION-
THE SPEED IS BETWEEN 10,000 RPM-20,000 RPM
• LOW SPEED CENTRIFUGATION- THE SPEED IS BELOW 10,000 RPM
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DIFFERENTIAL DIFFERENTIAL CENTRIFUGATIONCENTRIFUGATION
• 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
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DIFFERENTIAL DIFFERENTIAL CENTRIFUGATIONCENTRIFUGATION
• SUPERNATANT CONTAINS MATERIALS THAT ARE NOT SEDIMENTED BUT CAN BE SEDIMENTED WHEN CENTRIFUGATION IS DONE AT A HIGHER SPEED
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DIFFERENTIAL DIFFERENTIAL CENTRIFUGATIONCENTRIFUGATION
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ZONAL CENTRIFUGATIONZONAL 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)
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RATE-ZONAL RATE-ZONAL CENTRIFUGATIONCENTRIFUGATION
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ISOPYCNICISOPYCNIC--ZONAL ZONAL CENTRIFUGATIONCENTRIFUGATION
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SEDIMENTATION COEFFICIENTSEDIMENTATION 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
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SEDIMENTATION COEFFICIENTSEDIMENTATION COEFFICIENTVALUESVALUES
PARTICLE OR SEDIMENTATION
MOLECULE COEFFICIENT
LYSOSOME 9400S
TOBACCO MOSAIC VIRUS 198S
RIBOSOME 80S
RIBOSOMAL RNA MOLECULE 28S
tRNA MOLECULE 4S
HEMOGLOBIN MOLECULE 4.5S
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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)
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SPEED OF CENTRIFUGATION
• 2 WAYS OF EXPRESSING THE PULLING FORCE:
a) RELATIVE CENTRIFUGATIONAL FORCE-RCF (g)
b) ROTATION PER MINUTE (rpm)
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RELATIVE RELATIVE CENTRIFUGATIONAL FORCECENTRIFUGATIONAL 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
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RELATIVE RELATIVE CENTRIFUGATIONAL FORCECENTRIFUGATIONAL FORCE
• EQUATION
R.C.F. = 1.119 x 10 -5 (rpm2) r rpm=rotation per minute
r=radius (in cm)
UNIT g
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ELECTROPHORESISELECTROPHORESIS
• 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
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ELECTROPHORESISELECTROPHORESIS• 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 INTER-WOVEN MATRIX THAT ACTS LIKE A SIEVE TO SEAPARATE THE MOLECULE ACCORDING TO THEIR SIZE
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ELECTROPHORESISELECTROPHORESIS• 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
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PROTEIN ELECTROPHORESIS 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
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2-D PROTEIN 2-D PROTEIN ELECTROPHORESIS 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
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2-D PROTEIN 2-D PROTEIN ELECTROPHORESISELECTROPHORESIS
• 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
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2-D PROTEIN 2-D PROTEIN ELECTROPHORESISELECTROPHORESIS
• 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
+
-
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2-D PROTEIN 2-D PROTEIN ELECTROPHORESISELECTROPHORESIS
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1-D PROTEIN 1-D PROTEIN ELECTROPHORESISELECTROPHORESIS
• 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
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SDS-PAGESDS-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)
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SDS-PAGESDS-PAGE• PROTEIN IS STAINED USING COOMASIE
BLUE OR SILVER• NON-DIRECTIONAL STAINING CAN BE
DONE:
-ANTIBODY BOUND WITH RADIOISOTOPE OR ENZYME, FLUORESENCE DYE
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SDS-PAGESDS-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 LIPIDS
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SDS-PAGESDS-PAGE -MERCAPTOETHANOL FUNCTION: TO
BREAK DISULPHIDE BONDS SO THAT PROTEIN SUBUNIT CAN BE ANALYSED
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NUCLEIC ACID NUCLEIC ACID ELECTROPHORESISELECTROPHORESIS
• 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
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NUCLEIC ACID NUCLEIC ACID ELECTROPHORESISELECTROPHORESIS
• 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
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NUCLEIC ACID NUCLEIC ACID ELECTROPHORESISELECTROPHORESIS
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MICROSCOPYMICROSCOPY
• 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-100m)
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LIGHT MICROSCOPELIGHT 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
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LIGHT MICROSCOPELIGHT MICROSCOPE
• TYPES OF LIGHT MICROSCOPE :
BRIGHT-FIELD MICROSCOPE DARK-FIELD MICROSCOPE
PHASE-CONTRAST MICROSCOPE
FLUORESENCE MICROSCOPE (UV)
(FLUORESCIN/RHODAMIN)
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ELECTRON MICROSCOPEELECTRON 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
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ELECTRON MICROSCOPEELECTRON 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
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ELECTRON MICROSCOPEELECTRON 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
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ELECTRON MICROSCOPEELECTRON MICROSCOPE
SCANNING ELECTRON MICROSCOPE
MOSQUITO IMAGESBY SCANNING ELECTRONMICROSCOPE
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OTHER TECHNIQUESOTHER TECHNIQUES
• CHROMATOGRAPHY
-PAPER: PROTEIN SEPARATION BY USING FILTER PAPER AS THE MATRIX
-ION-EXCHANGE
-GEL FILTRATION
-AFFINITY
-HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)
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OTHER TECHNIQUESOTHER 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