For SDS-PAGE [second-dimensional; vertical polyacrylamide slab gel; 190 x 180 x 1 mm]
2-D gel slab electrophoresis chamber unit (Anatech).
Accessories:
Immobiline Dry Strip supporting plate (Pharmacia Code No. 18-1004-34)
Strip fixing coumb (Anatech).
The source of electric power:
Programmable Power Supply Model 3800 (Anatech)
2-D gel analysis and databasing:
PDQUEST software package, (PDI Version 5.1.2)
Reagents and Consumables:
Electrofocusing sample application pieces, (Pharmacia Code No. 80-1129-46)
Electrofocusing electrode strips, (Pharmacia Code No. 18-1004-40)
Silver stain kit (Wako Code No. 299-13841)
Silcon oil 5c/s (Sinetsu Chem. Code No. KF-96-L-5CS)
2D-protein pI marker (Daiichi Code No. 220760)
Protein Mr marker (Daiichi Code No. 181122 and 181066)
Gel:
For the first-dimensional isoelectric focusing:
Immobiline Drystrips, pH 4-7 or pH 3-10 NL, 18 cm long (Pharmacia), are re-hydrated overnight in Gel Swelling Solution as follows (quantities are for 8 strips):
urea 19.2 g
10%(v/v) Triton X-100 2.0 ml
Pharmalyte 3-10 0.2 ml
0.1 M acetic acid 1.0 ml
dithiothreitol 80 mg
0.1%(w/v) Orange 1.0 ml
pure water up to 40 ml
For the second-dimensional SDS-PAGE:
7.5%T, 3%C polyacrylamide gel mix is prepared as follows (quantities are for 8 slabs, 190 x 180 x 1 mm):
35 %(w/v) acrylamide 100 ml
2 %(w/v) BIS-acrylamide 55 ml
1.5 M Tris-HCl, pH 8.8 117 ml
50%(v/v) glycerol 80 ml
pure water up to 470 ml
--- degass in vacuo before adding below---
10 %(w/v) SDS 4.8 ml
10 %(w/v) ammonium persulfate 0.5 ml
TEMED 0.3 ml
Procedure of protein extraction from cell cultures:
- Rinse the culture dish with 5 ml of PBS- for 1 min, repeat 3 times.
- Scrape off cells from the culture dish on ice by rubbing with a plastic scraper.
- Transfer the cell suspension to a 1.5-ml microfuge tube of which tare was weighed in advance. (Give W [mg]= the weight of the cell suspension)
- Add W x 0.85 mg of urea, W x 0.05 micro-liter of 2-mercapoethanol, W x 0.02 micro-liter of 1.5%(w/v) SDS, 8.5%(v/v) Triton X-100.
- Apply ultrasonic power for 1 sec x 10 times. In the case of TAITEC VP-5S, set output control = 6.
- Centrifugation at 15,000 rpm for 20 min at room temperature.
- Store small aliquots of the supernatant below -70C until use for electrophoresis. (Incubate for 3 min at 45C prior to application)
Procedure of isoelectric focusing:
- Pour a small volume of silicon oil into the electrophoresis chamber, mount Immobiline DryStrip supporting plate in the chamber giving care to remove air bubbles beneath the plate.
- Add 1 micro-liter of pI marker protein solution to an aliquot (10-20 micro-liter) of cell extract if neccessary. (In the case of "DAIICHI" 2D pI Marker for silver staining, the content of a vial is dissolved in 0.5 ml of pure water. The reconstituted solution is storable at -20C ).
- Stand re-swelled Immobiline Dry Strip on its long edge on filterpaper briefly to remove excess Gel Swelling Solution, then lay on the filterpaper gel-side-up.
- Put a piece of Sample Application Filter on the gel ca. 1-cm inside from the cathodic end. Apply the cell extract (10-20 micro-liter) to the Sample Application Filter.
- Mount the strip in the electrophoresis chamber which is pre-cooled to 20C.
- Put electrode pads wetted with distilled water on both ends of the strip to recieve sure contact of platinum electrodes.
- Set the platimum electrode, and cover the gel strip and electrode pads with silicon oil (5 c/s) to isolate electrophoresis media from the atmosphere.
- Apply electric power under the programed control as follows:
Step Voltage Time (min)
1 500 V 180 min
2 700 V 60 min
3 1000 V 60 min
4 1500 V 60 min
5 2000 V 60 min
6 2500 V 60 min
7 3000 V 720 min
Keep the electric power supply at 500 V after step 7. (Avoid a prolonged standing at 500 V)
SDS-treatment of gel strip
Incubate for 40 min at room temperature in fresh SDS-treatment solution prepared as below (the quantity is for 8 strips).
SDS-treatment solution:
urea 29.0 g
dithiothreitol 0.4 g
0.5 M Tris-HCl, pH 6.8 8.0 ml
10%(w/v) SDS stock @ 16.0 ml
0.1%(w/v) BPB 2.0 ml
50%(v/v) glycerol 40.0 ml
After incubation, wrap the strip with a Saran Wrap and store below -70 C until proceed to SDS-PAGE
Procedure of SDS-PAGE
- Pour an appropriate volume of the anodic electrode solution into the vertical electrophoresis chamber.
- Incubate the gel strip in SDS-treatment solution for 5 min at room temperature again.
- Fill the top of the gel slab with the cathode solution, place the gel strip that is trimmed to fit the gel size, and gently push the strip down using a shark tooth-shaped Strip Fixer Comb to achieve the firm contact to the gel slab.
- Mount the gel plate on the electrophoresis chamber unit.
- Fill the cathodic chamber with the cathodic electrode buffer.
- Perform electresis in 10 mA/slab until the BPB dye comes near the gel bottom.
- Proceed to the subsequent protein staining or an electro-transfer blotting.
Buffer Stock Solutions are prepared as follows:
10x concentrated stock of anodic electrode buffer (2 M Tris-HCl, pH 8.8):
dissolve
Tris 242.2 g
in 700 ml of Milli-Q SP water.
Adjust pH to 8.8 with 6N HCl
Add pure water upto 1,000 ml
5x concentrated stock of cathodic electrode buffer (Tris-Tricine Buffer ):
dissolve
Tris 60.5 g
Tricine 89.5 g
SDS 5.0 g
in 1,000 ml of pure water.
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