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:
- PageRuler Plus
- Gal4EED-luc: H340_MV11 + g034_pSPgRNA
- Gal4EED-luc: H340_MV11 (no gRNA)
- Gal4EED-luc: H360_MV11 + g034_pSPgRNA
- Gal4EED-luc: H360_MV11 (no gRNA)
- Gal4EED-luc: H380_MV11 + g034_pSPgRNA
- Gal4EED-luc: H380_MV11 (no gRNA)
- dummy
- dummy
- dummy
- dummy
- 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)
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