• 00:18 1.
    Western Blot - 3_Semi-Dry transfer [Video from GeneTex]
  • 00:12 2.
    Transfer proteins to nitrocellulose membrane
  • 00:08 2.1
    equilibrate in transfer buffer for 15 min
  • 00:13 2.2
    Rinse 4 pieces of blot paper with transfer buffer
  • 00:09 2.3
    Place 2 pieces of pre-soaked blot paper onto the platinum anode
  • 00:02 2.4
    Roll a test tube over the layer to remove bubbles
  • 00:08 2.5
    Place the pre-wetted nitrocellulose membrane on top of the wetted blot paper
  • 00:08 2.6
    Place the equilibrated gel on top of the nitrocellulose membrane
  • 00:04 2.7
    Place 2 pieces of pre-wetted blot paper on top of the gel
  • 00:03 2.8
    Roll a test tube over the layer to remove bubbles
  • 00:11 2.9
    Sop up transfer buffer with paper towel
  • 00:08 2.10
    Place the cathode plate onto the stack
  • 00:04 2.11
    Place safely cover on the unit
  • 00:16 2.12
    Run the transfer unit at 18V for 1 h
  • 00:06 2.13
    Rinse the membrane with H2O
  • 00:00 3.
    Check for success of transfer
  • 00:17 3.1
    Stain the nitrocellulose with Ponceau Red solution
  • 00:10 3.2
    Wash in H2O unter the water is clear and protein bands are well-defined
  • 00:11 3.3
    The membrane can be destained by washing in TBST or water
  • 00:00 4.
    Block the membrane
  • 00:06 4.1
    Incubate the membrane in blocking buffer at RT for 30-60 min
  • 00:01 4.2
    Pour off the blocking buffer
  • 00:00 5.
    Incubate with the primary antibody
  • 00:09 5.1
    Incubate the membrane in blocking buffer with primary antibody at 4C overnight
  • 00:00 6.
    Wash the nitrocellulose membrane
  • 00:03 6.1
    Pour off the primary antibody solution
  • 00:13 6.2
    Wash the membrane 5 times for 3-5 min each time with TBST
  • 00:00 7.
    Incubate with the secondary antibody
  • 00:15 7.1
    Incubate the membrane in blocking buffer with 2nd antibody at RT for 1 h
  • 00:00 8.
    Wash the nitrocellulose membrane
  • 00:03 8.1
    Pour off the 2nd antibody solution
  • 00:15 8.2
    Wash the membrane 5 times for 3-5 min each time with TBST
  • 00:00 9.
    Detect the proteins using detection system
  • 00:06 9.1
    Remove excess TBST
  • 00:04 9.2
    Follow the instruction of ECL kit
  • 00:15 9.3
    Mix equal vol. of Enhanced Luminol Reagent (A) and the Oxidizing Reagent (B)
  • 00:08 9.4
    Pour the chemiluminescence reagent into the membrane (incubate 1 min at RT)
  • 00:21 9.5
    Take a picture with CCD camera
  • Index
  • Notes
  • Comment
  • Fullscreen
Western Blot - 3_Semi-Dry transfer [Video from GeneTex]
Duration: 05:05, Browse: 763, Last Updated: 2018-12-20
Keypoint
  1. 1.
    Transfer proteins to nitrocellulose membrane
    .
  2. 2.
    Transfer proteins to nitrocellulose membrane
    .
  3. 3.
    Check for success of transfer
    .
  4. 4.
    Check for success of transfer
    .
  5. 5.
    Block the membrane
    .
  6. 6.
    Wash the nitrocellulose membrane
    .
  7. 7.
    Incubate with the secondary antibody
    .
    Prev
    • 00:18 1.
      Western Blot - 3_Semi-Dry transfer [Video from GeneTex]
    • 00:12 2.
      Transfer proteins to nitrocellulose membrane
    • 00:08 2.1
      equilibrate in transfer buffer for 15 min
    • 00:13 2.2
      Rinse 4 pieces of blot paper with transfer buffer
    • 00:09 2.3
      Place 2 pieces of pre-soaked blot paper onto the platinum anode
    • 00:02 2.4
      Roll a test tube over the layer to remove bubbles
    • 00:08 2.5
      Place the pre-wetted nitrocellulose membrane on top of the wetted blot paper
    • 00:08 2.6
      Place the equilibrated gel on top of the nitrocellulose membrane
    • 00:04 2.7
      Place 2 pieces of pre-wetted blot paper on top of the gel
    • 00:03 2.8
      Roll a test tube over the layer to remove bubbles
    • 00:11 2.9
      Sop up transfer buffer with paper towel
    • 00:08 2.10
      Place the cathode plate onto the stack
    • 00:04 2.11
      Place safely cover on the unit
    • 00:16 2.12
      Run the transfer unit at 18V for 1 h
    • 00:06 2.13
      Rinse the membrane with H2O
    • 00:00 3.
      Check for success of transfer
    • 00:17 3.1
      Stain the nitrocellulose with Ponceau Red solution
    • 00:10 3.2
      Wash in H2O unter the water is clear and protein bands are well-defined
    • 00:11 3.3
      The membrane can be destained by washing in TBST or water
    • 00:00 4.
      Block the membrane
    • 00:06 4.1
      Incubate the membrane in blocking buffer at RT for 30-60 min
    • 00:01 4.2
      Pour off the blocking buffer
    • 00:00 5.
      Incubate with the primary antibody
    • 00:09 5.1
      Incubate the membrane in blocking buffer with primary antibody at 4C overnight
    • 00:00 6.
      Wash the nitrocellulose membrane
    • 00:03 6.1
      Pour off the primary antibody solution
    • 00:13 6.2
      Wash the membrane 5 times for 3-5 min each time with TBST
    • 00:00 7.
      Incubate with the secondary antibody
    • 00:15 7.1
      Incubate the membrane in blocking buffer with 2nd antibody at RT for 1 h
    • 00:00 8.
      Wash the nitrocellulose membrane
    • 00:03 8.1
      Pour off the 2nd antibody solution
    • 00:15 8.2
      Wash the membrane 5 times for 3-5 min each time with TBST
    • 00:00 9.
      Detect the proteins using detection system
    • 00:06 9.1
      Remove excess TBST
    • 00:04 9.2
      Follow the instruction of ECL kit
    • 00:15 9.3
      Mix equal vol. of Enhanced Luminol Reagent (A) and the Oxidizing Reagent (B)
    • 00:08 9.4
      Pour the chemiluminescence reagent into the membrane (incubate 1 min at RT)
    • 00:21 9.5
      Take a picture with CCD camera
    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 18:19:29
    Last Updated
    2018-12-20 08:02:15
    Browse
    763
    Duration
    05:05
    Reference
    1