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    Lab 10 Electrophoresis

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    Erica Sujung Choe
    This document provides instructions for separating and visualizing proteins from food samples using electrophoresis. Key steps include: extracting proteins from foods using a saline buffer, separating extracted proteins by SDS-PAGE based on size, and estimating protein molecular weights by comparing migration distances to standards of known weight. The procedure allows comparison of protein patterns between food types to detect substitutions or differences in quality.

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    Lab 10 Electrophoresis

    Uploaded by

    Erica Sujung Choe
    140 views4 pages
    This document provides instructions for separating and visualizing proteins from food samples using electrophoresis. Key steps include: extracting proteins from foods using a saline buffer, separating extracted proteins by SDS-PAGE based on size, and estimating protein molecular weights by comparing migration distances to standards of known weight. The procedure allows comparison of protein patterns between food types to detect substitutions or differences in quality.

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    This document provides instructions for separating and visualizing proteins from food samples using electrophoresis. Key steps include: extracting proteins from foods using a saline buffer, separating extracted proteins by SDS-PAGE based on size, and estimating protein molecular weights by comparing migration distances to standards of known weight. The procedure allows comparison of protein patterns between food types to detect substitutions or differences in quality.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    140 views4 pages

    Lab 10 Electrophoresis

    Uploaded by

    Erica Sujung Choe
    This document provides instructions for separating and visualizing proteins from food samples using electrophoresis. Key steps include: extracting proteins from foods using a saline buffer, separating extracted proteins by SDS-PAGE based on size, and estimating protein molecular weights by comparing migration distances to standards of known weight. The procedure allows comparison of protein patterns between food types to detect substitutions or differences in quality.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 4

    FDSC 4050 LABORATORY

    PROTEIN SEPARATION BY ELECTROPHORESIS


    INTRODUCTION

    BACKGROUND
    Electrophoresis can be used to separate and visualize proteins. In sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), proteins are separated based on size. When protein samples are applied to such gels, it is usually necessary to know the protein content of the sample. This makes it possible to apply a volume of sample to the gel so samples have a comparable amount of total protein. While it is possible to use an official method of protein analysis (Kjeldahl, N combustion) for such application, it often is convenient to use a rapid analysis that requires only a small amount of sample. In this experiment, proteins are extracted with a salt solution, and the proteins in the extracts are separated and visualized by SDS-PAGE. The visualization of the proteins makes it possible to distinguish different types of foods because most foods have a characteristic protein pattern. For example, one can detect the substitution of inexpensive fish for expensive fish.

    OBJECTIVE
    Extract proteins from foods and separate and compare the proteins by electrophoresis.

    PRINCIPLE OF THE METHOD


    Proteins present in saline extracts can be separated by SDS-PAGE, which gives size separation. Proteins bind SDS to become negatively charged, so they move through the gel matrix toward the anode (pole with positive charge) at a rate based on size alone, since all molecules are highly negatively charged. The molecular weight of a given protein can be estimated by comparing its electrophoretic mobility with proteins of known molecular weight. A linear relationship is obtained if the logarithm of the molecular weights of standard proteins are plotted against their respective electrophoretic mobilities (Rf).

    CHEMICALS
    Sodium Chloride (NaCl) CAS No. 7647-14-5 Sodium phosphate, monobasic (NaH2PO4H2O) CAS No. 7558-80-7 Irritant Irritant

    REAGENTS

    SAMPLE EXTRACTION
    Extraction buffer Buffer of 0.15 M sodium chloride, 0.05 M sodium phosphate, pH 7.0

    SUPPLIES

    SAMPLE EXTRACTION
    Beaker, 250 ml Centrifuge tubes, 1.5 ml Cutting board Erlenmeyer flask, 125 ml Graduated cylinder, 50 ml Filter paper, Whatman No. 1 Food sample Funnel Knife Pasteur pipettes and bulbs Test tube with cap Weighing boat

    ELECTROPHORESIS
    Beaker, 250 ml (for boiling samples) Mechanical pipettors and tips 4-12% Bis-Tris gel Molecular weight marker

    EQUIPMENT
    Top loading balance Analytical balance Blender Centrifuge Electrophoresis unit Vortex mixer Water bath

    PROCEDURE
    (Single sample extracted)

    SAMPLE PREPARATION
    1. Coarsely cut up 100 g of food sample with a knife and accurately weigh out 90 g on a top loading balance (alternatively, weigh out the corresponding amount of freeze-dried sample and adjust with water or sample buffer).

    2. Blend 1 part sample with 3 parts extraction buffer (90 g food and 270 ml extraction buffer) for 1 min in a blender (Note: Smaller amounts of food and buffer, but in the same 1:3 ratio, can be used for a small blender). 3. Pour 1 ml of the food homogenate into each of two 1.5 ml centrifuge tubes. Label tube. Balance your tube against classmates sample. Use a spatula or Pasteur pipette to adjust tubes to an equal weight. 4. Centrifuge samples at 2000 x g for 15 min at room temperature. Collect the supernatant taking care not to collect any of the bottom particles or top layer of fat. Collect about 1 ml supernatant in a new 1.5 ml centrifuge tube. 5. Using the information in the label of your sample and comparing to it to the concentrations of samples from other groups, adjust the protein concentration of your sample to the lowest protein concentration among the different groups. 6. Show your calculations to the instructors before moving to the next step.

    ELECTROPHORESIS
    1. Assemble the electrophoresis unit according to manufacturers instructions. Position the gel(s) in the electrophoresis unit. 2. Mix the food extracts well, then dilute you sample with 4x sample buffer in an eppendorf tube. Make 40 l total. Mix well using the vortex, then open the tube. 3. Heat the samples for 3 min in a boiling water bath. Cool the sample and spin at high speed for about 12 seconds. 4. Load 20 L of your sample to the well of a 4-12% Bis-Tris gel. Also load 10 L of molecular weight marker. 5. Run the electrophoretic separation as directed by the TA. 6. At the end of the run, remove the gel and stain following the instructions from gels manufacturer. 7. Measure the migration distance (cm) from the top of the gel to the center of the protein band for the molecular weight standards and for each of the major protein bands in the food extract samples. 8. Observe and record the relative intensity of the major protein bands for each food extract. DATA AND CALCULATIONS 1. Calculate the relative mobility of three major protein bands and all the molecular weight standards. To determine the relative mobility (Rf) of a protein, divide its migration distance from the top of the gel to the center of the protein band by the migration distance of the last protein band on the molecular weight marker from the top of the gel

    Distance of Protein Migration Molecular weight standards 1 2 3 4 5 Food Sample Sample Identity

    Distance of Last Protein Migration

    Relative Mobility

    Molecular Weight

    2. Prepare a standard curve by plotting relative mobility (x-axis) versus log molecular weight of standards (y-axis). 3. Using the standard curve, estimate the molecular weight of the major proteins in the food extracts. QUESTIONS Your lab report should be short, concise, and should include a picture of your gel. Answer the following questions. 1. Describe how you would prepare 1 L of the buffer used to extract the food proteins (0.15 M sodium chloride, 0.05 M sodium phosphate, pH 7.0). Show all calculations 2. Based on the MW markers, identify the proteins in your samples and provide each protein a nominal MW. 3. Tentatively, which major protein(s) may be present in your sample? 4. Can you separate a 700 Da peptide using the gel that we used in the lab? Why?

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