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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 l 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-100m) 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