Group 2 - SDS-PAGE

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  • SDS-PAGE (SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS) BIOE 402 Principles of Molecular Biology

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  • OVERVIEW

    • What is SDS-PAGE?

    • Materials/Equipment Required in SDS-PAGE

    • Procedure

    • Applications of SDS-PAGE

    • Advantages of SDS-PAGE

    • Limitations of SDS-PAGE

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  • WHAT IS SDS-PAGE?

    • An electrophoretic system developed by Ulrich K. Laemmli

    • Used to separate proteins based on their molecular weight

    • Used to separate proteins of complex mixtures

    • Widely used in forensics, genetics, biotechnology, and molecular biology

    • Proteins are separated in polyacrylamide gels

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  • FUNCTION OF SDS IN SDS-PAGE

    • SDS is a detergent with a strong protein-denaturing effect

    • SDS binds to the protein backbone at a constant molar ratio

    • Proteins unfold into linear chains with negative charge in the presence of SDS

    • Proteins are separated based on polypeptide chain length

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  • PRINCIPLES OF SDS-PAGE

    • Smaller proteins migrate faster through the gel matrix

    • Sodium dodecyl sulfate and polyacrylamide gel eliminate the influence of structure and charge

    • Proteins are separated based on polypeptide chain length

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  • PRINCIPLES OF SDS-PAGE

    • Proteins are separated based on polypeptide chain length

    • Proteins are folded with positive and negative charges

    • Disulfide bonds are reduced

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      • PROCEDURE Electrophoresis

        • Turn on the power supply

        • Run the gel until the dye (BPB) in the sample buffer reaches the bottom of the gel

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      • PROCEDURE Electrophoresis

        • Remove the gel assembly from the electrophoresis apparatus

        • Remove the gel from the glass plates using a spatula

        • Prepare for subsequent analysis

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      • APPLICATIONS OF SDS-PAGE

        • Serves as a versatile tool for molecular weight measurement

        • Facilitates the estimation of protein size

        • Plays an important role in peptide mapping

        • Supports comparative analysis on the polypeptide composition of different structures

        • Indispensable for purity estimation

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      • APPLICATIONS OF SDS-PAGE

        • Used in Western Blotting and protein ubiquitination

        • Used in HIV test to separate the HIV proteins

        • Helps to analyze the size and number of polypeptide subunits

        • Used to analyze post-translational modifications

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      • ADVANTAGES OF SDS-PAGE

        • Helps determine the molecular weight of protein in a sample

        • Can detect specific proteins based on their characteristics and mass

        • Can identify different species based on the mass of the protein

        • Provides high resolution electrophoretic separation

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      • LIMITATIONS OF SDS-PAGE

        • Limited separation based on molecular weight

        • Denaturation of proteins

        • Inaccurate size determination

        • Sample handling and preparation time-consuming

        • Smaller or larger proteins may not separate well or may migrate unpredictably

        • Denaturation of proteins by SDS disrupts native protein structures

        • Migration of proteins through the gel can lead to inaccuracies in estimating molecular weight

        • Contaminants, incomplete denaturation, or improper loading can lead to inaccurate results

        • Time-consuming technique, especially when multiple samples need to be analyzed