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    Chemical

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    aksahu01234
    The document provides an overview of electrophoresis, including: 1) Electrophoresis is a technique that separates charged particles using an electric field. 2) It is used to separate macromolecules like DNA, RNA, and proteins based on size and charge. 3) Factors like electric field strength, pH, size and shape of molecules influence electrophoretic mobility and separation. 4) Common applications include separation of nucleic acids, proteins, amino acids, and analysis in forensics and clinical settings.

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    Chemical

    Uploaded by

    aksahu01234
    11 views27 pages
    The document provides an overview of electrophoresis, including: 1) Electrophoresis is a technique that separates charged particles using an electric field. 2) It is used to separate macromolecules like DNA, RNA, and proteins based on size and charge. 3) Factors like electric field strength, pH, size and shape of molecules influence electrophoretic mobility and separation. 4) Common applications include separation of nucleic acids, proteins, amino acids, and analysis in forensics and clinical settings.

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    The document provides an overview of electrophoresis, including: 1) Electrophoresis is a technique that separates charged particles using an electric field. 2) It is used to separate macromolecules like DNA, RNA, and proteins based on size and charge. 3) Factors like electric field strength, pH, size and shape of molecules influence electrophoretic mobility and separation. 4) Common applications include separation of nucleic acids, proteins, amino acids, and analysis in forensics and clinical settings.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    11 views27 pages

    Chemical

    Uploaded by

    aksahu01234
    The document provides an overview of electrophoresis, including: 1) Electrophoresis is a technique that separates charged particles using an electric field. 2) It is used to separate macromolecules like DNA, RNA, and proteins based on size and charge. 3) Factors like electric field strength, pH, size and shape of molecules influence electrophoretic mobility and separation. 4) Common applications include separation of nucleic acids, proteins, amino acids, and analysis in forensics and clinical settings.

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    © All Rights Reserved
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    Download as pptx, pdf, or txt
    of 27

    SANT GURUGHSIDAS GOVT.

    PG COLLEGE
    KURUD DISTRIC -DHAMTARI
    DEPARTMENT OF ZOOLOGY
    PAPER - 4
    FIRST SEMESTER SEESION - 2023

    GUIDED BY…….. SUBMITED BY…….

    H.O.D. Mr. Hit Narayan Tandan sir Chandraprabha sahu


    Msc. Zoology 1st semester
    ASS. Prof Chitramani shrimali mam
    WEL - COME
    CONTENTS

    • Introduction
    • definition
    • History
    • Principal
    • Effective Factor
    • Types
    • Application
    • Advantage & disadvantage
    • Conclusion
    • Refrence
    INTRODUCTION

    • Electrophoresis is process of migration of charged particle through a solution under the


    influence of external electric field.
    • Electrophoresis is used in laboratories to saperate macromolecules based on size.
    • Electrophoresis is used extensively in DNA, RNA and Protein analysis.
    DEFINITION

    • Electrophoresis is separation techniques based on movement of change particle in on


    electric field .
    • Electrophoresis is a technique in which charge molecule migrate to opposite charge.
    • Migration of molecule is depend on shape and size or charge.
    HISTORY

    • Father of electrophoresis Arne Tiselius in 1930 developed moving boundary


    electrophoresis.
    PRINCIPAL

    • When change particle place in electric field then positive change molecule travel to negative ions and
    negative change molecule travel to positive ions .
    • Migration is depend on shape,size and charge
    • V=eq/f

    • V = velocity
    • e = electricity
    • q =molecule change
    • f = fractions
    EFFECTIVE FACTOR

    • ELECTRIC FIELD- Separation of molecule is depend on electric field which depends on

    current voltage.
    if increase the rate of current then increase the separation rate of molecule
    pH OF MEDIUM – if any reason change the pH then rate of separation increase or
    decrease .
    SIZE AND SHAPE OF MOLECULE- if increase the size of molecule then decrease the
    separation rate.
    Free Electrophoresis:

    In this type of electrophoresis a free electro­lyte is taken in place of supporting media.


    Nowadays this type of electrophoresis has be­come out-dated and mostly used in non-
    biological experiments.

    It is mostly of two types―

    1.The micro-electrophoresis- which is mostly used in calculation of Zeta potentials


    (a colloidal prop­erty of cells in a liquid medium) of the cells.

    2.Moving boundary electrophoresis-which for many years had been used for
    quantitative analysis of complex mixtures of macromol­ecules, especially proteins.
    GEL ELECTROPHORESIS

    • Definition - Gel electrophoresis is a method used for separation and analysis of


    macromolecules (DNA,RNA & protein) & their fragments based on their size and
    charge.
    • Principal - Gel electrophoresis is based on the separation of DNA & their fragments
    through help of gel under the influence of electric field.
    • (-)ve change anion move forward anode & (+)ve change cation move forward cathode.
    TYPE OF GEL

    1. Starch gel – This type of gel is used horizontal zone electrophoresis. Gel are prepared
    by heating or cooling of partially hydrolysed Starch in a proper buffer solution.
    2. Agarose/ agar gel - Gels have fairly large pore size and are used for separating lager
    DNA molecules (Restrictions Fragment Length Polymorphism Analysis).
    3. Polyacrylamide – Gels are used to obtain high resolution separation for smaller DNA
    molecules (STR analysis and DNA sequence analysis).
    VISUALIZATION

    • The molecules in the gel are stained to make them visible. DNA may be visualised using
    ethidium bromide which, when intercalated into DNA, fluoresce under ultraviolet light,
    while protein may be visualised using silver stain or coomassie Brilliant Blue dye.
    • SYBR Green I is more expensive, but 25 time more sensitive and possible Safer than
    ethidium bromide.
    • SYBR safe is a variant of SYBR green and show low level of mutagenicity and toxicity.
    • Other less frequently used marker are cresol red and orange G.
    PAPER ELECTROPHORESIS

    • Introduction – The technique of paper electrophoresis is simple and inexpensive and


    required only micro quantities of plasma for separation.
    • The support medium is a filter paper.
    • The electrophoresis apparatus in its simplest form consist of two through to contains
    buffer solution, through which electric current is passed.
    • Frequently used in isolation proteins, amino acids and oligonucleotides.
    WORKING

    1. A long strip of filter paper is moistened with a suitable buffer solution of the desired pH and
    the sample is applied transversely across the central part of strip.
    2. End are fixed to dip in buffer solution in two through fitted with electrodes.
    3. Electric field of about 20 volts/cm is stabilized.
    4. The charge particles of sample Migrate a long strip towards respective electrodes of opposite
    polarity, according to net charges, size and introductions with the solid matrix.
    5. Homologous group of particles migrate as a separate band.
    6. The electrophoresis is carried out for 16 – 18 hours.
    OBSERVATION

    • The different fractions appear as blue coloured band across the Filter paper starting from
    the moving boundary backwards.
    • If a quantitative estimation is required for each fraction the bands may be carefully cut
    and eluted, or the bands may be scanned optically in a densitometer.
    • In human plasma five different bands can be identified on paper electrophoresis.
    CAPILLARY ELECTROPHORESIS
    Definition- an analytical technique that separates ions based on their electrophoresis mobility with
    the use of an applied voltage.

    Principal - Separation is based on the different electrophoretic mobility of analytes (μ e), which
    under an electric field (E) migrate as sharp zones at different velocity (v = μ eE).

    Isotachophoresis
    The technique of isotachophoresis depends on the development of potential gradient.
    Electro osmotic flow

    The surface of the silicate glass capillary contains negatively charged functional groups that
    attract positively-charged counter ions. The positively-charged ions migrate towards the
    negative electrode and carry solvent molecules in the same direction. This overall solvent
    movement is called electro osmotic flow. During a separation, uncharged molecules move at
    the same velocity as the electro osmotic flow (with very little separation). Positively-charged
    ions move faster and negatively-charged ions move slower.

    A small volume of sample is moved into one end of the capillary. The capillary passes through
    a detector, (usually a UV absorbance detector), at the opposite end of the capillary.

    • Application of a voltage causes movement of sample ions towards their appropriate electrode
    usually passing through the detector.

    • A plot of detector response with time is generated which is termed an electropherogram


    APPLICATION
    1. Separation of Deoxyribonucleic acid
    2. Separation of ribonucleic acid
    3. Separation of protein molecules
    4. It may be used as preparative technique prior to use methods such as mass spectroscopy, cloning,
    DNA Southern blotting for further characterization.
    5. Separation of amino acid
    6. Separation of lipoproteins
    7. Separation of enzyme in blood
    8. Separation of antibiotic drug
    ADVANTAGE AND DISADVANTAGE

    Advantages:
    • (1) simple and inexpensive apparatus that permits simultaneous analysis of several
    samples in a relatively routine procedure.
    • (2) simple procedures for visualization of zones and for isolation of fractions.
    Disadvantages:
    • gels can melt during electrophoresis, the buffer can become exhausted, and different
    forms of genetic material may run in unpredictable forms.
    CONCLUSION

    Electrophoresis can be considered as an electro kinetic phenomenon involving an electric


    field, supporting medium, and a buffer solution. It has many forensic and clinical
    applications in our daily life.

    Reference- book - tools and tachniqe


    writer - S.M. Saxena.
    THANK YOU
    (1) Which technique separates charged particles using electric field?
    [A] Hydrolysis
    [B] Electrophoresis
    [C] Protein synthesis
    [D] Protein denaturing

    The electrophoretic mobility denoted as µ is mathematically


    expressed as:
    [A] VE
    [B] V/E
    [C] E/V
    [D] 1/EM

    Which of the following factors does not influence


    electrophoretic mobility?
    [A] Molecular weight
    [B] Shape of molecule
    [C] Size of molecule
    [D] Stereochemistry of molecule
    When is electrophoresis not used?
    [A] Separation of proteins
    [B] Separation of amino acids
    [C] Separation of Lipids
    [D] Separation of nucleic acids

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