Plan

title: Plan updated: 2024-02-23T13:24:49 created: 2024-01-10T09:47:34

Plan středa 10. ledna 2024 9:47

• Seed
• Transfect
• Fix, wwash, block, primary
• Secondary
• Cells: ~TREX 293~ HEK293T
• Plasmids
  • Notch1-HA
  • GSK3B-FLAG
    • Also GSK3B-HA?
  • VDAC1-FLAG
  • pcDNA3.1
  • Bluescript
• Samples
  • WT
  • Notch1 OE
  • GSK3B OE
  • VDAC1 OE
  • N1 + GSK3B
  • N1 + VDAC
  • (GSK3B + VDAC)
  • N1 + GSK3B + VDAC
• Abs
  • Anti-N1
  • Anti-HA
  • Anti-FLAG
• Mito-tracker - while seeding
• 24 well plate
  • Gelatin
• Day 1
  • Seed cells, gelatin, 24 well plate, cover slips
  • 60 - 100k cells /well
• Day 2
  • Transfection
• Day 3
  • Remove medium
  • Add mitotracker
  • Wash
  • Fix - formaldehyde
  • Wash
  • Block
  • Primary ab 4 °C ON
• Day 4
  • Wash
  • Secondary ab 1 h RT
  • Wash
  • Dapi/hoechst
  • PBS, mounting media

| | | | | --- | --- | --- | | Ha (ms) | 488 | 647 | | FLAG (rb) | 594 | 488 | | Mitotracker | | 599 | | I'll | | |

| Sample | N1 | GSK3B | VDAC1 | pcDNA | BS | 1 Ab | Mitotracker | | --- | --- | --- | --- | --- | --- | --- | --- | | WT (N1) | - | - | - | 150 Ng | 250 ng | Anti—N1 | + | | N1-HA | 50 ng | - | - | 100 ng | 250 ng | ~Anit-N1,~ anti-HA | + | | GSK3B-FLAG | - | 50 ng | - | 100 ng | 250 ng | ~Anti-N1,~ anti-FLAG | + | | VDAC1-FLAG | - | - | 50 ng | 100 ng | 250 ng | ~Anti-N1,~ anti-FLAG | + | | N1-HA + GSK-FLAG | 50 ng | 50 ng | - | - | 250 ng | ~Anti-N1,~ anti-FLAG, anti-HA | - | | N1-HA + VDAC-FLAG | 50 ng | - | 50 ng | 50 ng | 250 ng | ~Anti-N1,~ anti-FLAG, anti-HA | - | | N1-HA + VDAC-FLAG + GSK-FLAG | 50 ng | 50 ng | 50 ng | - | 250 ng | ~Anti-N1,~ anti-HA, (anti-flag) | + | | GSK3B-HA + VDAC-FLAG | - | 50 ng (HA) | 50 ng | 50 ng | 250 ng | ~Anti-N1~, anti-flag, anti-HA | - | | N1-HA + GSK-FLAG | 50 ng | 50 ng | - | - | 250 ng | ~Anti-N1,~ , anti-HA | + | | N1-HA + VDAC-FLAG | 50 ng | - | 50 ng | 50 ng | 250 ng | ~Anti-N1,~ anti-FLAG, anti-HA | + |