• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Preparation of Brain Cytosol

        互联网

        760

         

        Preparation of Brain Cytosol


        1. Chill a glass plate on ice.

        2. Place brain tissue on the chilled glass plate and cut slices of brain into small pieces.

        3. Resuspend the pieces in ice-cold RIPA buffer without the detergents NP-40 and Na deoxycholate. Use 1L per 250 g of tissue

        4. Perform the following step in a cold room : Transfer the suspended tissue to a blender and homogenize on low power with four 30 second bursts. Let the homogenate cool for one minute between bursts. Repeat bursts until the homogenate is smooth.

        5. Transfer the homogenate to pre-chilled centrifuge tubes.

        6. Centrifuge at 2900 x g for 20 minutes at 4�. This step sediments unbroken cells and nuclei. Discard the pellet.

        7. Transfer the supernatant fraction to another centrifuge tube.

        8. Centrifuge this supernatant fraction at 29,000 x g for 45 minutes at 4�.

        9. Transfer and save the supernatant fraction. This fraction contains brain cytosol and microsomal membranes. Concentrate the brain cytosol proteins by precipitation with glacial acetic acid as described below.

        10. Resuspend the pellet in 10 ml of ice-cold RIPA buffer containing detergents (please see protocol "Preparation of Modified RIPA Buffer").

        11. Centrifuge the resuspended pellet at 15,000 x g for 20 minutes at 4�.

        12. Transfer the supernatant fraction to another tube. This fraction contains solubilized mitochondrial and plasma membrane proteins.

        Concentration of Brain Cytosolic Proteins by Precipitation with Glacial Acetic Acid

        CAUTION: Glacial acetic acid is caustic. Perform the following step in a hood and wear gloves, goggles, and a lab coat.

        1. Acidify the brain cytosol fraction (Step 9 above) to pH 4.5 by slowly adding and mixing drops of glacial acetic acid.

        2. Sediment the precipitated proteins by centrifuging the acidified sample at 15,000 x g for 20 minutes at 4�. Discard the supernatant fraction.

        3. Dissolve the pellet in 10 ml of desired buffer.

        4. Determine protein concentration by a standard method.

         

        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序