User:Karmella Haynes/Notebook/PcTF Genomics/2015/08/18: Difference between revisions
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==08/18/15== | ==08/18/15== | ||
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* Western - Castone PAGE & blot | * Western - Castone PAGE & blot, Stain (anti-HA tag | ||
---- | ---- | ||
'''Western - Castone PAGE & blot'''<br> | '''Western - Castone PAGE & blot, Stain'''<br> | ||
* 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 | * Troubleshooting: saw some indication of signal last time; BSA is recommended for modified proteins | ||
** Re-run the Gal4-EED sample set | ** 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) | ||
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# dummy | # dummy | ||
Run gel: 120 V, | Run gel: 120 V, 90 min. | ||
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RESULTS | 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) | |||
Revision as of 20:03, 18 August 2015
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08/18/15
Western - Castone PAGE & blot, Stain
General:
Protein sample prep:
Loading:
Run gel: 120 V, 90 min.
Electroblot
Poncaeu stain
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