Matt:LabNotes/Polyacrylamide Gel Protocol
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Polyacrylamide Gel Protocol
Purpose and General Procedure
- Make gel to cover and mechanically stabilize brain tissue section for DARTFISH to prevent tissue degradation
- Instead of using dessicated bind-silane treated slides we have to use vectabond-treated cover glass in a tissue culture dish with a hydrated tissue section
- Due to thickness of culture dish (~1mm) no way to use coverslip to prevent exposure to air while making <1mm thick gel
- Can try using mineral oil to cover instead
- Due to thickness of culture dish (~1mm) no way to use coverslip to prevent exposure to air while making <1mm thick gel
- Not using DATD (cross-linker) in Acrylamide/Bis mix
Materials
- 10% BSA (Sigma Alrich Website
- 40% Acrylamide
- 40% Acrylamide/Bis (19:1)
- Syringe and 0.22 micron filter
- Ammonium persulfate (APS)
- TEMED
- Dessicator
Protocol
- Turn on UV lamp in AirClean hood for 15 minutes.
- Prepare fresh "AB mix" in a 0.5 ml pcr tube:
- 90 uL IEF 40% Acrylamide (made from powder)
- 10 uL Acrylamide / Bis (19:1; 38%:2%)
- Using a 3-cc syringe and a 0.22 micron filter, filter ~200 uL of the ABD mix into a 1.5 mL microcentrifuge tube.
- Prepare fresh 5% APS (ammonium persulfate). Notably, the APS bottle should be stored in some sort of room-temperature dessicator. We just store it inside a large plastic screw-top container.
- 5 mg APS →100 uL dH20
- Prepare fresh 5% TEMED (in fume hood)
- 2 uL TEMED → 38 uL dH20.
- Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C)
- Prepare the gel-casting mix (200 uL total volume). Do not add APS until immedietely prior to casting the gels. This recipe is for a 10% gel with sufficient mix for at least 8 (identical) slides.
- 50 uL AB mix (FILTERED)
- 4 uL 10% BSA
- 138 uL dH20
- 4 uL 5% TEMED
- 4 uL 5% APS
- Add