User:Karmella Haynes/Notebook/PcTF Genomics/2015/08/18

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08/18/15

  • Western - Castone PAGE & blot, Stain (anti-HA tag)



Western - Castone PAGE & blot, Stain

  • Detect dCas fusion via the C-terminal HA tag: https://benchling.com/s/8fVChs2U/edit
  • Troubleshooting: saw some indication of signal last time; BSA is recommended for modified proteins
    • Re-run the Gal4-EED sample set
    • 5% milk/PBST blocking buffer should be fine for the HA tag (no post-translational modification, large protein)


SDS-PAGE

General:

Protein sample prep:

  • Final volume = 20 μL
  • Sample buffer: 4x Nupage LDS sample buffer
  • 4x sample master mix (x13)
    • 13x 5.0 μL 4x LDS = 65
    • 13x 1.0 μL 1M DTT = 13
  • Protein samples (6): 14 μL protein + 6 μL master mix
  • Dummy samples (5): 14 μL water + 6 μL master mix
  • Heat @ 100°C/ 5 min.Cool at room temp.

Loading:

  1. PageRuler Plus
  2. Gal4EED-luc: H340_MV11 + g034_pSPgRNA
  3. Gal4EED-luc: H340_MV11 (no gRNA)
  4. Gal4EED-luc: H360_MV11 + g034_pSPgRNA
  5. Gal4EED-luc: H360_MV11 (no gRNA)
  6. Gal4EED-luc: H380_MV11 + g034_pSPgRNA
  7. Gal4EED-luc: H380_MV11 (no gRNA)
  8. dummy
  9. dummy
  10. dummy
  11. dummy
  12. dummy

Run gel: 120 V, 90 min.


TRANSFER

Electroblot

  • Load both at same time, "feet" facing inward
  • Transblot Turbo transfer pack, mini format, 0.2 um PVDF
  • Bio-Rad Transblot Turbo: Bio-Rad Protocol, Mixed MW, 2.5A, 25V, 7 min (slot A, upper)

Poncaeu stain

  • Rinsed blot in di-water and wahed in 1x Poncaeu for ~15 min.
  • Rinsed off excess with di-water


RESULTS

  • Saw a 10 kD band this time...gel needs to run longer for proteins to reach the resolving end of the gel
  • Used PVDF membrane (ordered a mixed nitro/PVDF Turbo-blot pack by accident...?)
  • PVDF is hard to stain with Poncaeu (reported on various websites)
  • However, PVDF gave much better signal in the previous trial than nitrocellulose
  • Cut away dummy lanes and extra membrane from bottom
  • Continue with primary stain


PRIMARY STAIN

  • Block 1 hour at RT in 5% milk/ PBST
  • New floating chamber
    • made one from a rectangular PCR tube rack with tight-fitting lid (Bioexpress, GeneMate R-7909-2)
    • platform: cut an emptied pipette tip cartridge (flat plastic) in half to roughly fit the membrane
    • wrapped 96-well PCR plate sealing film over the platform to cover up holes (prevent pits from forming)
    • covered platform with parafilm (did not stretch it too much) for clean, disposable surface
  • Antibody: 1:500 poly rabbit anti-HA (500 μL)
  • Laid membrane face-down onto diluted primary
  • Covered with lid, sealed with parafilm
  • Incubated overnight at 4°C (cold room)