• Home
  • Biopharmaceutical Research Services
  • Multi-Omics Services
  • Support
  • /assets/images/icon/icon-email-2.png

    Email:

    info@MtoZ-Biolabs.com

    How to Prepare Protein Samples for SDS-PAGE?

      Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a fundamental technique used to separate proteins based on their molecular weight. This paper outlines the detailed steps required for the preparation of protein samples for SDS-PAGE, emphasizing the importance of each step in ensuring accurate and reproducible results.

       

      SDS-PAGE is a widely used analytical method in molecular biology and biochemistry for the separation of proteins. Proper preparation of protein samples is critical to achieving high-resolution and reproducible results. This paper provides a comprehensive guide to preparing protein samples for SDS-PAGE, covering sample collection, lysis, quantification, and preparation of the sample buffer.

       

      Materials and Methods

      1. Sample Collection and Storage

      (1) Collect the protein sample using appropriate methods, such as tissue homogenization or cell lysis.

      (2) Store samples at -80°C to prevent protein degradation until further processing.

       

      2. Sample Lysis

      (1) Use a lysis buffer containing Tris-HCl, NaCl, EDTA, and protease inhibitors to lyse cells or tissues. The exact composition of the lysis buffer should be optimized based on the specific protein source, ensuring effective lysis and protein solubilization.

      (2) Sonicate the sample on ice to ensure complete cell disruption and homogenization, minimizing proteolytic activity.

       

      3. Protein Quantification

      (1) Quantify the protein concentration using a Bradford or BCA protein assay to ensure consistent loading of protein samples onto the gel.

      (2) Standardize the protein concentration across all samples to ensure equal protein amounts are loaded onto the gel.

       

      4. Preparation of Sample Buffer

      (1) Prepare the sample buffer by mixing Tris-HCl (to maintain pH), SDS (to denature proteins), glycerol (to add density), bromophenol blue (to visualize the sample during loading), and β-mercaptoethanol (as a reducing agent to break disulfide bonds).

      (2) The β-mercaptoethanol ensures proteins are fully denatured by reducing disulfide bonds.

       

      5. Sample Heating

      (1) Mix equal volumes of the protein sample and sample buffer.

      (2) Heat the mixture at 95°C for 5 minutes to ensure complete protein denaturation.

       

      6. Loading the Gel

      (1) Load the denatured protein samples onto the SDS-PAGE gel carefully to avoid cross-contamination between wells.

      (2) Ensure equal amounts of protein are loaded into each well to allow accurate comparison across samples.

       

      7. Electrophoresis

      (1) Run the gel at a constant voltage (typically 100-150V) until the dye front reaches the bottom of the gel.

      (2) Use a molecular weight marker to determine the size of the separated proteins accurately.

       

      The preparation of protein samples for SDS-PAGE involves several critical steps that must be meticulously followed to achieve reliable results. Proper sample collection, lysis, quantification, and denaturation are essential for the accurate separation and analysis of proteins by SDS-PAGE.

    Submit Inquiry
    Name *
    Email Address *
    Phone Number
    Inquiry Project
    Project Description *

     

    How to order?


    /assets/images/icon/icon-message.png

    Submit Inquiry

    /assets/images/icon/icon-return.png