BIOCHEMICAL TECHNIQUES:: PART 7

ELECTROPHORESIS

INTRODUCTION

-         This is a technique that is used to separate charged molecules based on their mobility in electric field.

-         This technique is widely used in the separation of DNA.

                                     

-         Mobility depends upon the net charge of the molecule, size of the molecule, shape of the molecule and strength of the magnetic field.

BIOCHEMICAL TECHNIQUES:: PART 6

Characteristics of beads

-         Chemically inert.

-         Uniform pore and particle size.

-         High mechanical rigidity.

-         Contains no ionic groups.

Advantages of gel penetration

-         Gentleness.

-         Maximum recovery of sample.

BIOCHEMICAL TECHNIQUES:: PART 5

GEL PENETRATION

INTRODUCTION

-         This method uses gel beads that are made of inert material and have pores in them.

-         This method is used when molecules are to be separated based on their size.

-         The particles with larger size run down fast as they cannot get into pores of the beads, since smaller particles get into the pores of the beads they take longer time to finish the run.

BIOCHEMICAL TECHNIQUES:: PART 4

Types of Centrifugation Based On Speed (RPM)

-         Low speed centrifugation

o   RPM is between 3000-6000.

o   RCF is 6000g.

o   Large organelles like nucleus and chloroplast are separated.

-         Micro speed centrifugation

o   RPM is 12000.

o   RCF is 10000g.

-         High speed centrifugation

o   RPM is 25000.

o   RCF is 60000g.

o   Used to separate mitochondria and lysosomes.

-         Ultracentrifugation

o   RPM is 30000.

o   RCF id 60000g.

o   Used to separate ribosomes, vesicles and other biological macromolecules.

BIOCHEMICAL TECHNIQUES:: PART 3

Types of Centrifugation Based On Density 

-          RATE ZONAL

o    The separation of molecules is based on the sedimentation coefficient of the molecule.

                  

-         ISOPYCNIC

o   Separation on basis of buoyant density.

        

-         DENSITY BARRIER

                     

BIOCHEMICAL TECHNIQUES:: PART 2

CENTRIFUGATION

INTRODUCTION

-         This method of separation is based on sedimentation rate.

-         Sedimentation rate depends upon the size and shape of the molecule and the viscosity of the solution.

-         Higher the molecular weight greater the rate of sedimentation.

-         In centrifugation we rotate a suspension at a very high speed so that the components settle down quickly.

                              

-         When a suspension is centrifuged the components of the suspension of the suspension are separated into pallets and supernatant.

-         Every suspension has to be centrifuged at a specific speed for a specific time for its separation.

BIOCHEMICAL TECHNIQUES:: PART 1

Introduction

-         These methods are used by biologists, biotechnologists, genetic engineers, pharmacologists and others for the separation of one kind of biomolecules from other.

-         Specific techniques are used for separation of specific molecules.

-         These techniques exploit the physical, chemical and electrical properties of molecules for their separation.

                     

-           These include a large number of techniques like centrifugation, chromatography, gel penetration techniques, electrophoresis, spectrometry and many more.