Dicty Flag-Tag Western Blot Protocol

Notes on the starting Dicty cells.The volume of a 24-well microtiter plate well is 0.5 ml minus evaporation. Number of cells to load on gel is 5x10^5. A confluent Dicty plate is about 5 x 10^6/mL. (The depth of media on the plate or well is kept constant, so it is normal to mix up area and volume when referring to cell density.)

Things to do in advance

  1. Freeze cooler block from gel transfer device
  2. Check supply of gel transfer buffer in cold room.

Prepare Gel Sample from Dicty Cells (~1 hour)

  1. Dicty expressing (we hope) the locus 5 construct have grown up in a 24 well microtiter plate well. Janet estimates that the cell density is 5 x 10^6 cells per ml.
  2. Run a gel (12% in this case since construct is large). Use an untransformed control.
  3. To see the protein use about 5 x 10^6 cells, or 1/5 of the well in this case. Janet is taking 1/5 to preserve the clone, and will give me 4/5 of the well content. In theory I would load about 1/4 of this on the gel, but some cells were lost so I will load 1/2.
  4. Spin down the cells for 5 min at 680 g (2900 rpm on our ufuge). Carefully aspirate media.
  5. Resuspend (by gently pipetting up and down) in 20 uL of TE and boil immediately to minimize degradation. Boil for 5 minutes.
  6. Add an equal volume of 2x sample buffer and boil 5 more min.
  7. Load 20 ul onto gel. Load 10 ul of the control cells!

Transfer to nitrocellulose paper (~1 hour).

  1. Find apparatus (BIO RAD Mini Trans Blot Cell). Freeze cooling block beforehand.
  2. Transfer buffer should be made and chilled in advance. Make more if you use it up. It is SDS protein running buffer with 20% MeOH.
  3. Get out 2 small trays, fill one with water and the other with buffer. Add buffer at the last minute so its cool. Avoid bubbles in buffer as they block xfer.
  4. Trim your gel down and notch the lower left corner.
  5. Trim nitrocellulose paper (BIO-RAD cat # 162-0116, 0.45 uM) to a bit bigger than your gel. Wet in water.
  6. Make a sandwich with the black side of the swiss-cheese clamp ending up on the bottom. Sponge. Blotter paper. Gel (flip gel over at this point). Nitrocellulose paper. Blotter paper. Sponge. Clamp sandwich together. Be careful not to include bubbles.
  7. Insert sandwich in the tray so the black side of the sandwich is toward the black terminal, the idea being to run-to-red from gel to nitrocellulose. Fill with buffer part way, insert cooler block and add more buffer so it is over the top edge of the paper.
  8. Run on high-amp power supply in cold room at 100 V (constant V) for 1 hour. 0.31 amps is a typical current.

Blocking and Binding Primary Ab (~ 1 hour of work + O/N incubation).

  1. Prepare in advance blocking/Ab-binding buffer: 200 mL 2% milk in 1X TBS. Prepare a few minutes in advance and stir. PONSO stain BEFORE adding this stuff to the NC paper.
  2. PONSO Stain the NC paper. Find a square petri dish and lid. Trace appropriate segments of the gel outline on the NC paper with a #2 or softer pencil. Peel gel off and discard. Rinse the SDS buffer off of the NC paper (interferes w/ PONSO). In the square petri dish add enough PONSO stain to cover the NC paper. Staining is fast since protein is on surface. Pour the PONSO back in the bottle. Make a photocopy by putting NC paper in a good sheet protector. Trace the MW marker bands with a #2 or softer pencil (or use prestained markers).
  3. Add about 10 mL Blocking/Binding buffer and rock for 1 hour at RT.
  4. Add 2.5 ul of · mouse monoclonal antiflag Ab, borrowed from Jennifer in the Coluccio lab (in this case) to 10 mL of Blocking/Binding buffer (a 5000-fold dilution in this case). Rock in cold room overnight. Note: this is where you want to be at the end of the day).

Wash, bind secondary Ab, final wash (~ 2 hours).

  1. Wash 3x in milk buffer for 10 min. @RT.
  2. Add 10 uL (this case) of secondary Ab with 10 ml milk buffer (1000x dilution) at RT for an hour (· antimouse Ab Ab linked with horse radish peroxidase, borrowed from Vlad in Sherman lab).
  3. Wash 3x for 10 min.
  4. Rinse w/ TBS until clear.

ECL

  1. Find reagents and materials for ECL: Peirce kit in the fridge door. 1 mL pipetman. Tweezer. Fresh square microtiter plate. Film. Film cassette. Sheet protector marked with phosphorescent ink. Fine point sharpie. Timer.
  2. Let the NC paper drip dry and transfer it to the new tray.
  3. Pipet 1 mil of each ECL reagent to a corner of the tray, away from the gel. Mix by pipetting onto the gel. Time for 5 min. with occasional pipetting up and down.
  4. Pick up the NC paper with the tweezer and let the reagent drip off. Transfer it to the sheet protector.
  5. Load film and gel in darkroom and expose for 1 minute.
  6. Run film through the processor. When film emerges use the pencil markings on the NC paper to mark the film (lanes, MW bands, cut-off corner of gel, etc.).
  7. Make 10 min. and 10 sec. exposures too.