Techniques in molecular biology
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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

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TECHNIQUES IN MOLECULAR BIOLOGY

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Techniques in molecular biology

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

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.html


Analytical centrifugation

ANALYTICAL CENTRIFUGATION

  • IS USED TO MEASURE THE SEDIMENTED PARTICLE PHYSICAL CHARACTERISTICS SUCH AS SEDIMENTATION COEFFICIENT AND MOLECULAR WEIGHT


Preparative centrifugation

PREPARATIVE CENTRIFUGATION

  • TO SEPARATE SPECIFIC PARTICLES THAT IS REUSABLE

  • TYPES:

    - RATE ZONAL

    - DIFFERENTIAL

    - ISOPYCNIC CENTRIFUGATION


Ultracentrifugation and low speed

ULTRACENTRIFUGATION AND LOW 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


Differential centrifugation

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 centrifugation1

DIFFERENTIAL CENTRIFUGATION

  • SUPERNATANT CONTAINS MATERIALS THAT ARE NOT SEDIMENTED BUT CAN BE SEDIMENTED WHEN CENTRIFUGATION IS DONE AT A HIGHER SPEED


Differential centrifugation2

DIFFERENTIAL CENTRIFUGATION


Zonal 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

RATE-ZONAL CENTRIFUGATION


Isopycnic zonal centrifugation

ISOPYCNIC-ZONAL CENTRIFUGATION


Sedimentation coefficient

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

SEDIMENTATION COEFFICIENTVALUES

PARTICLE OR SEDIMENTATION

MOLECULE COEFFICIENT

LYSOSOME9400S

TOBACCO MOSAIC VIRUS198S

RIBOSOME80S

RIBOSOMAL RNA MOLECULE 28S

tRNA MOLECULE4S

HEMOGLOBIN MOLECULE 4.5S


Speed of centrifugation

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 centrifugation1

SPEED OF CENTRIFUGATION

  • 2 WAYS OF EXPRESSING THE PULLING FORCE:

    a) RELATIVE CENTRIFUGATIONAL FORCE-RCF (g)

    b) ROTATION PER MINUTE (rpm)


Relative centrifugational force

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 force1

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

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


Electrophoresis1

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 INTER-WOVEN MATRIX THAT ACTS LIKE A SIEVE TO SEAPARATE THE MOLECULE ACCORDING TO THEIR SIZE


Electrophoresis2

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 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

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 electrophoresis1

+BASIC

-ACIDIC

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.


2 d protein electrophoresis2

+

-

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 electrophoresis3

2-D PROTEIN ELECTROPHORESIS


1 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 PRESENTDURINGSAMPLE PREPARATION

  • SIMULATION OF 1-D ELECTROPHORESIS

    http://www.rit.edu/~pac8612/electro/

    Electro_Sim.html


Sds page

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 page1

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 page2

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 LIPIDS


Sds page3

SDS-PAGE

  • -MERCAPTOETHANOL FUNCTION: TO BREAK DISULPHIDE BONDS SO THAT PROTEIN SUBUNIT CAN BE ANALYSED


Nucleic acid electrophoresis

NUCLEIC ACID ELECTROPHORESIS

  • AGAROSE OR POLIACRYLAMIDE IS THEMATRIX 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 electrophoresis1

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 electrophoresis2

NUCLEIC ACID ELECTROPHORESIS


Microscopy

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

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 microscope1

LIGHT MICROSCOPE

  • TYPES OF LIGHT MICROSCOPE :

    BRIGHT-FIELD MICROSCOPE DARK-FIELD MICROSCOPE

    PHASE-CONTRAST MICROSCOPE

    FLUORESENCE MICROSCOPE (UV)

    (FLUORESCIN/RHODAMIN)


Electron microscope

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 microscope1

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 microscope2

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 microscope3

ELECTRON MICROSCOPE

SCANNING ELECTRON

MICROSCOPE

MOSQUITO IMAGES

BY SCANNING ELECTRON

MICROSCOPE


Other techniques

OTHER TECHNIQUES

  • CHROMATOGRAPHY

    -PAPER: PROTEIN SEPARATION BY USING FILTER PAPER AS THE MATRIX

    -ION-EXCHANGE

    -GEL FILTRATION

    -AFFINITY

    -HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)


Other techniques1

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


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