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    Lecture 6 DP Protein Seperation

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    AkshayaaRavee
    SDS-PAGE is a technique used to separate proteins by molecular weight through gel electrophoresis, where proteins treated with SDS migrate through a polyacrylamide gel matrix and are visualized using protein stains with differing sensitivities such as Coomassie Blue, SYPRO Orange, or silver stain to detect protein bands of varying quantities down to nanogram levels. The process involves denaturing proteins with SDS and reducing agents, running them through stacking and resolving gels with an applied electric current, and staining to view band migration based on molecular size.

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    Lecture 6 DP Protein Seperation

    Uploaded by

    AkshayaaRavee
    30 views23 pages
    SDS-PAGE is a technique used to separate proteins by molecular weight through gel electrophoresis, where proteins treated with SDS migrate through a polyacrylamide gel matrix and are visualized using protein stains with differing sensitivities such as Coomassie Blue, SYPRO Orange, or silver stain to detect protein bands of varying quantities down to nanogram levels. The process involves denaturing proteins with SDS and reducing agents, running them through stacking and resolving gels with an applied electric current, and staining to view band migration based on molecular size.

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    SDS-PAGE is a technique used to separate proteins by molecular weight through gel electrophoresis, where proteins treated with SDS migrate through a polyacrylamide gel matrix and are visualized using protein stains with differing sensitivities such as Coomassie Blue, SYPRO Orange, or silver stain to detect protein bands of varying quantities down to nanogram levels. The process involves denaturing proteins with SDS and reducing agents, running them through stacking and resolving gels with an applied electric current, and staining to view band migration based on molecular size.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    30 views23 pages

    Lecture 6 DP Protein Seperation

    Uploaded by

    AkshayaaRavee
    SDS-PAGE is a technique used to separate proteins by molecular weight through gel electrophoresis, where proteins treated with SDS migrate through a polyacrylamide gel matrix and are visualized using protein stains with differing sensitivities such as Coomassie Blue, SYPRO Orange, or silver stain to detect protein bands of varying quantities down to nanogram levels. The process involves denaturing proteins with SDS and reducing agents, running them through stacking and resolving gels with an applied electric current, and staining to view band migration based on molecular size.

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    © All Rights Reserved
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    of 23

    The SDS-PAGE

    Dr Suresh
    What is SDS-PAGE?

    SDS-PAGE
    (= sodium dodecylsulphate-
    polyacrylamide gel
    electrophoresis)

    -method for separation of


    proteins according to their
    molecular weight
    SDS PAGE is a technique for separating proteins based
    on their molecular weight.

    Large proteins migrate slowly through the gel matrix and


    small proteins migrate quickly through the matrix

    The porous gel matrix is composed


    of the polymer polyacrylamide
    Link

    polymerization Cross Link

    The polymerization reaction is catalyzed by


    TEMED (tetramethyl Ethylenediamine and
    APS (ammonium persulfate)
    PAGE

     Gels are cast by polymerizing a solution of acrylamide


    monomers into polyacrylamide chains
     Gel pore size can be varied by adjusting the concentrations
    of polyacrylamide
     Smaller proteins migrate faster than larger proteins through
    the gel
    Native proteins
    SDS (sodium dodecyl sulfate) binds to and coat the
    protein
    The hydrophobic region of SDS binds to the
    hydrophobic region of proteins and unfolds them.

    This also covers the proteins with a negative charge.


    All proteins now have a net negative charge.

    -----------------------------
    SDS
    -----------------------------
    SDS

     all polypeptide chains are then forced into extended


    conformations
     SDS treatment eliminates the effect of differences in shape
     individual polypeptide chains migrate as a negatively
    charged SDS-protein complex through the porous
    polyacrylamide gel
     speed of migration is proportional to the size of the proteins
     smaller polypeptides running faster than larger polypeptides
    How about covalent link?

    DTT/Me SH

    S-S

    HS
    Disulfide bonds must be broken to
    completely unfold the protein.
    Reducing agents/antioxidents are used to break the disulfide bonds.
    DTT: Dithiothreitol
    and
    BME: Beta mercaptoethanol - - - -
    - -
    S S -
    -
    SDS -
    S ------------------- S -
    ----------------------- - -

    S -----------------------------
    SDS
    S -----------------------------
    Plus DTT
    Protein loading buffer
     Protein gel loading buffer contains Tris buffer to maintain
    constant pH
     glycerol to increase sample density,
     the strong ionic detergent SDS (sodium dodecylsulfate),
     β-mercaptoethanol, a reducing agent. . Beta-mercaptoethanol
    eliminates disulfide bonds in proteins by reducing them (adding
    hydrogen atoms).
     Heating
    -proteins denatured by heating them
    in a sample loading buffer containing
    sodium dodecyl sulphate (SDS)

    -the proteins no longer have any


    secondary, tertiary or quaternary
    structure
    -resultant proteins take on a rod-like
    shape and a uniform negative charge-
    to-mass ratio proportional to their
    molecular weights
    Negatively charged protein samples are
    loaded into wells at the top of the gel

    An electric current is applied so that there is a


    positive charge at the bottom of the gel and a
    negative charge at the top of the gel.

    The negatively charged proteins will migrate


    to the bottom of the gel according to size.
    Stacking gel
     To obtain optimal resolution of proteins, a “stacking” gel is
    poured over the top of the “resolving” gel.
     The stacking gel
    lower concentration of acrylamide (larger pore size),
    lower pH
    different ionic content
     This allows the proteins in a lane to be concentrated into a
    tight band before entering the running or resolving gel
     produces a gel with tighter or better separated protein bands
    Visualizing the proteins:
    Types of Stains:
    1. Coomassie Blue – Sensitivity of 0.1ug protein per band

    Traditional method requires staining followed by destaining to remove


    background gel staining.
    GelCode Blue from Pierce does NOT stain the gel and requires no
    destaining. Increased sensitivity of 25ng protein per band

    2. Silver Stain – Sensitivity of 2ng protein per band

    Glassware and plasticware must be very clean to prevent stain from


    precipitating out of solution.

    Prone to high background due to impurities in the acrylamide and


    surface artifacts such as fingerprints
    Lane 1. Kaleidoscope Markers
    2. Shark
    3. Salmon
    4. Trout
    5. Catfish
    6. Sturgeon
    7. Actin and Myosin Standard
    Comparison of the sensitivity of
    SDS PAGE stains:
    Identical SDS-polyacrylamide gels
    were stained with

    A) SYPRO Orange protein gel stain


    B) SYPRO Red protein gel stain
    C) silver stain
    D) Coomassie brilliant blue stain
    Outline of the experiment
    *Prepare polyacrylamide gels
    *Add diluted samples to the sample buffer
    *Heat to 95C for 4 minutes
    *Load the samples onto polyacrylamide gel
    *Run 200 volts for 30-40 minutes
    *Stain in Coomassie Blue stain
    *Destain
    *Identify molecular markers, proteins

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