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    Membraneand Cytosol Preparation MOD

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    Fadia Kamal
    This document provides instructions for preparing membrane and cytosol fractions from mouse heart tissue. The procedure involves homogenizing the crushed heart tissue, centrifuging to separate the nuclei and debris from the supernatant containing membrane and cytosol fractions, then ultracentrifuging to pellet the membrane fraction. The supernatant is saved as the cytosolic fraction and the membrane pellet is resuspended in binding buffer. Aliquots of both fractions are frozen at -80°C for storage.

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    Membraneand Cytosol Preparation MOD

    Uploaded by

    Fadia Kamal
    20 views2 pages
    This document provides instructions for preparing membrane and cytosol fractions from mouse heart tissue. The procedure involves homogenizing the crushed heart tissue, centrifuging to separate the nuclei and debris from the supernatant containing membrane and cytosol fractions, then ultracentrifuging to pellet the membrane fraction. The supernatant is saved as the cytosolic fraction and the membrane pellet is resuspended in binding buffer. Aliquots of both fractions are frozen at -80°C for storage.

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    MembraneandCytosolPreparationMOD

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    This document provides instructions for preparing membrane and cytosol fractions from mouse heart tissue. The procedure involves homogenizing the crushed heart tissue, centrifuging to separate the nuclei and debris from the supernatant containing membrane and cytosol fractions, then ultracentrifuging to pellet the membrane fraction. The supernatant is saved as the cytosolic fraction and the membrane pellet is resuspended in binding buffer. Aliquots of both fractions are frozen at -80°C for storage.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
    20 views2 pages

    Membraneand Cytosol Preparation MOD

    Uploaded by

    Fadia Kamal
    This document provides instructions for preparing membrane and cytosol fractions from mouse heart tissue. The procedure involves homogenizing the crushed heart tissue, centrifuging to separate the nuclei and debris from the supernatant containing membrane and cytosol fractions, then ultracentrifuging to pellet the membrane fraction. The supernatant is saved as the cytosolic fraction and the membrane pellet is resuspended in binding buffer. Aliquots of both fractions are frozen at -80°C for storage.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
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    Membrane and Cytosol Preparation

    Preparation Create 100mL stocks of Mouse Heart Lysis Buffer and Beta Binding Buffer; do not add protease inhibitor to stocks Create stock of protease inhibitor: Dissolve one tablet in 250uL of distilled H20; aliquot in 50uL to create a 50x stock. Add 20uL for every 1mL of buffer. Or dissolve one tablet in 10mL of buffer. Phosphatase inhibitors can be found in -20C. Prepare three 50mL conical tubes: put distilled H2O into two of them and ethanol into the 3rd Fill red tub with ice Clean homogenizer by running it first in 50mL conical with ethanol, then in the first 50mL conical with water, then in the second 50mL conical with water, and finally in a 15mL conical with ~1mL of Lysis Buffer. Save each of the buffers as controls. Procedure 1. To a 17x100mm tube on ice add 0.5 to 1ml of Mouse Heart Lysis Buffer. As a general rule it is best to add 1ml Mouse Heart Lysis Buffer for every 100 to 150mg of tissue. 2. Place the container with the heart into liquid nitrogen. When ready, using forceps, dip a few weighing dishes into liquid nitrogen to collect some of the liquid nitrogen and then place heart into this dish. Cover dish with another dish and then crush the heart with a hammer. 3. Use a scraper to place as much of the heart into the 17x100mm tube as possible. 4. Homogenize for 20 to 30sec or until you can no longer see large pieces of tissue. If the homogenate seems viscous you should add more Mouse Heart Lysis Buffer to add more volume. Fill small beaker with ice and place tube in beaker to keep sample cold. 5. Transfer sample to 1.7mL tube and spin sample at 800rcf for 15min at 4o C. - The pellet will be nuclei and cellular debris while the supernatant will contain the membrane and cytosol. 6. Save supernatant in new tube and repeat homogenization step for the pellet (if necessary). 7. Transfer supernatant to a clean tube. If the supernatant is cloudy it should be filtered through 4 layers of cheesecloth. The pellet from the 800rcf spin can be discarded. 8.Spin supernatants at 100,000xg (53,000rpm) for 1 hour at 4oC on the ultracentrifuge (in Taubman lab). 9. After the 100,000xg spin the supernatant should be pipetted off and frozen in the -80 C. (This is the cytosolic fraction). Make at least two aliquots. 10. Resuspend pellet (the membrane fraction) in Beta Binding Buffer. Use a 27G1/2 syringe to dislodge pellet. Add to 1.7ml snap cap tubes and freeze at -80 oC. Make two aliquots. You are trying to get an end concentration of 1mg/ ml for you membrane. Remember it is easier to dilute than concentrate.

    11. It may be necessary to concentrate the cytosol at the end of the prep. To do this we use Centricon centrifugal filter devices. Millipore has list of to help to determine the proper filter size for the protein of interest. Mouse Heart Lysis Buffer 25mm Tris 5mm EDTA 5mm EGTA pH 7.4 @ 37o C filter For 100mL 2.5mL Tris (1M) 1mL EDTA (0.5M) 5mL EGTA (100mM) 91.5mL dH2O

    + protease and phosphatase inhibitors Beta Binding Buffer 75 mm Tris 12.5 mm MgCl2 2 mm EDTA pH 7.4 @ 37o C filter + protease and phosphatase inhibitors

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