• 00:00 1.
    Western Blot - 1_Sample preparation [Video from GeneTex]
  • 00:18 2.
    Checking cells
  • 00:30 3.
    Remove medium
  • 00:43 4.
    Wash cells
  • 00:59 5.
    Lyse cells
  • 01:13 6.
    Scrape cells
  • 01:23 7.
    Collect cells
  • 01:33 8.
    Vortex
  • 01:39 9.
    Centrifuge
  • 01:41 9.1
    Balance the tubes
  • 01:54 10.
    Collect supernatant
  • 02:05 11.
    Denature
  • 02:23 11.1
    Boil the mixture at 95-100 C for 5 min
  • Index
  • Notes
  • Comment
  • Fullscreen
Western Blot - 1_Sample preparation [Video from GeneTex]
Duration: 02:33, Browse: 1213, Last Updated: 2018-12-19
Keypoint
  1. 1.
    Checking Cells
    Before collecting the cells, the cells should be checked microscoptically to ensure they are healthy and growing as expected.
  2. 2.
    RIPA buffer
    RIPA buffer
     
    Tris-HCl (pH 7.6)    25mM
    NaCl                       150 mM
    Triton X-100                1%
    Na-deoxychola            1%
    SDS                             1%
     
    Store at 4C
    Add the protease inhibitors immediately prior use
     
  3. 3.
    Centrifuge Tip
    Use the right size of tubes for the right centrifuge. If tube size is too small, you need to add adaptor.
  4. 4.
    Centrifuge
    20,000 g at 4C for 30 min
  5. 5.
    Denature
    6X SDS Sample Buffer:
    Tris-HCl (pH6.8)     315.5mM
    Glycerol               50%
    SDS                   0.1 g/ml
    bromophenol blue      0.05%
    2-b mercaptoethanol    10%
    Next
      1. Q1.
        When to add protease inhibitors?
      2. Q2.
        What is the setting of centrifuge?
      3. Q3.
        Collect the supernatant and place in a fresh tube kept on ___?
      4. Q4.
        What is the purpose of adding sample buffer?
      • 00:00 1.
        Western Blot - 1_Sample preparation [Video from GeneTex]
      • 00:18 2.
        Checking cells
      • 00:30 3.
        Remove medium
      • 00:43 4.
        Wash cells
      • 00:59 5.
        Lyse cells
      • 01:13 6.
        Scrape cells
      • 01:23 7.
        Collect cells
      • 01:33 8.
        Vortex
      • 01:39 9.
        Centrifuge
      • 01:41 9.1
        Balance the tubes
      • 01:54 10.
        Collect supernatant
      • 02:05 11.
        Denature
      • 02:23 11.1
        Boil the mixture at 95-100 C for 5 min
      Location
      1. Media Center
      2. ...
      3. Western blot (Genetex版本)
      Folder name
      Western blot (Genetex版本)
      Author
      賴亮全
      Branch
      賴亮全教授
      Created
      2018-12-19 18:18:30
      Last Updated
      2018-12-19 22:33:54
      Duration
      02:33
      Reference
      1

      臺北市仁愛路一段1號10樓1009室

      R1009 10F No1 Sec1 Ren-Ai Rd, Taipei 100233, Taiwan.

      E-mail: llai@ntu.edu.tw

      Tel: 886-2-2312-3456 # 288241

      Lab: 886-2-2393-8235