techniques in molecular biology centrifugation- separation of molecules/macromolecules/organelles...

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

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

ANALYTICAL CENTRIFUGATIONANALYTICAL CENTRIFUGATION

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

PREPARATIVE PREPARATIVE CENTRIFUGATIONCENTRIFUGATION

• TO SEPARATE SPECIFIC PARTICLES THAT IS REUSABLE

• TYPES:

- RATE ZONAL

- DIFFERENTIAL

- ISOPYCNIC CENTRIFUGATION

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

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

DIFFERENTIAL DIFFERENTIAL CENTRIFUGATIONCENTRIFUGATION

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

DIFFERENTIAL DIFFERENTIAL CENTRIFUGATIONCENTRIFUGATION

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)

RATE-ZONAL RATE-ZONAL CENTRIFUGATIONCENTRIFUGATION

ISOPYCNICISOPYCNIC--ZONAL ZONAL CENTRIFUGATIONCENTRIFUGATION

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

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

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 FORCE-RCF (g)

b) ROTATION PER MINUTE (rpm)

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

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

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

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

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

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 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

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

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

+

-

2-D PROTEIN 2-D PROTEIN ELECTROPHORESISELECTROPHORESIS

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

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)

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

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

SDS-PAGESDS-PAGE -MERCAPTOETHANOL FUNCTION: TO

BREAK DISULPHIDE BONDS SO THAT PROTEIN SUBUNIT CAN BE ANALYSED

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

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

NUCLEIC ACID NUCLEIC ACID ELECTROPHORESISELECTROPHORESIS

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)

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

LIGHT MICROSCOPELIGHT MICROSCOPE

• TYPES OF LIGHT MICROSCOPE :

BRIGHT-FIELD MICROSCOPE DARK-FIELD MICROSCOPE

PHASE-CONTRAST MICROSCOPE

FLUORESENCE MICROSCOPE (UV)

(FLUORESCIN/RHODAMIN)

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

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

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

ELECTRON MICROSCOPEELECTRON MICROSCOPE

SCANNING ELECTRON MICROSCOPE

MOSQUITO IMAGESBY SCANNING ELECTRONMICROSCOPE

OTHER TECHNIQUESOTHER TECHNIQUES

• CHROMATOGRAPHY

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

-ION-EXCHANGE

-GEL FILTRATION

-AFFINITY

-HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)

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