Matt:LabNotes/Polyacrylamide Gel Protocol

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Polyacrylamide Gel Protocol[edit]

Purpose and General Procedure[edit]

  • 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
  • Not using DATD (cross-linker) in Acrylamide/Bis mix

Materials[edit]

  • 10% BSA (Sigma Alrich Website
  • 40% Acrylamide
  • 40% Acrylamide/Bis (19:1)
  • Syringe and 0.22 micron filter
  • Ammonium persulfate (APS)
  • TEMED
  • Dessicator

Protocol[edit]

  1. Turn on UV lamp in AirClean hood for 15 minutes.
  2. Prepare fresh "AB mix" in a 0.5 ml pcr tube:
    1. 90 uL IEF 40% Acrylamide (made from powder)
    2. 10 uL Acrylamide / Bis (19:1; 38%:2%)
  3. 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.
  4. 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.
    1. 5 mg APS →100 uL dH20
  5. Prepare fresh 5% TEMED (in fume hood)
    1. 2 uL TEMED → 38 uL dH20.
  6. Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C)
  7. 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.
    1. 50 uL AB mix (FILTERED)
    2. 4 uL 10% BSA
    3. 138 uL dH20
    4. 4 uL 5% TEMED
    5. 4 uL 5% APS
  8. Add