Protocols

• Stain the gel 1h 30 - 2 h or overnight in coomassie blue R-250 solution (CBB R-250 0.1%, MeOH 50%, 10% acetic acid).
• Destain in destain solution: 10% acetic acid, 40% methanol in bidistilled water until desired background is obtained.
• Store the gel in 10% ethanol, at 4°C if for long times.

Preparation of cell lysate for 2D PAGE

• Pellet the cells in a suitable centrifuge at 4C
• Pour off all growth media
• Resuspend cell pellet in 1ml of cell wash buffer, transfer cells into a 1.5 ml tube
• Spin cells in a microcentrifuge 1-2 min at 200xg,
• Remove and discard the supernatant
• Repeat wash steps 4 times
• Ensure all the wash buffer has been removed using a fine pipette
• Resuspend quickly and uniformly the washed cell pellet in X volume (see below) of Lysis buffer (DLB) and leave on ice 10min
• Sonicate cells ( I do it 5x 5sec, and cool tubes on ice 1 min between sonication) with a tip sonicator
• Centrifuge the cell lysate 10min 13'000rpm, 4C
• Transfer supernatant into a new 1.5ml tube
• Repeat centrifugation step 1x
• Transfer supernatant into a new 1.5ml tube. This is the cell lysate to be used ;
• If possible make a protein quantification using the Bradford method, the concentration must be at least of 4-5ug/ul
• start 2D procedure immediately if possible. Otherwise snap-freeze in liquid N2 and keep at -80C

Cell Wash buffer 10mM Tris pH 8.0 5mM Magnesium Acetate Aliquot and store at -20C Lysis buffer (DLB) 30mM Tris 7M Urea 2M Thiourea 4% CHAPS Adjust to pH 8.5 with dilute HCL Aliquots and store at -20C For information For my cells I use 15.10⁵ cells in 300ul Lysis buffer, and got a concentration of 6ug/ul

More sensitive than regular Coomassie, but also longer to do.
According to Candiano et al. (Electrophoresis 25, 1327–1333, 2004).

Reagents

* orthophosphoric acid
* methanol
* Ammonium  sulfate
* Coomassie brilliant blue G-250

Preparation: for 1000 ml

* mix 100 ml ddH2O and 100 ml ortophosphoric acid
* add 100 g ammonium sulfate, stir to dissolve
* add 1.2 g Coomassie blue G-250
* add water to 800 ml total volume, stir
* add 200 ml methanol to a total volume of 1000 ml
* stir well, the solution is ready to use
* DO NOT FILTER, mix well the solution before use.

Staining :

* after migration wash gels in ddwater for 10 min
* add Coomassie solution and shake 4h to overnight for maximal staining
* variant : gels can be fixed 30 min in fixing solution, followed by a 20 min-wash in water and then Candiano staining. This is probably advisable for thick gels especially.

Adapted from Blum et al., Electrophoresis 8, 93-99, 1987

• Solutions:
• Solution A: prepare 100 ml of 50% MeOH, 10% acetic acid in bidistilled H2O (v/v).
• Solution B: prepare 100 ml of 5% MeOH in bidistilled H2O (v/v).
• Solution C: dissolve 0.2 g of sodium thiosulfate (Na2S2O3) in 1 l bidistilled H2O.
• Solution D: dissolve 200 mg of silver nitrate (AgNO3) in 100 ml bidistilled H2O.
• Solution E: mix 3 g sodium carbonate (Na2CO3), 50 µl of formaldehyde (HCOH 37%), 2 ml of solution C and complete to 100 ml with bidistilled H2O.
• Solution F: dissolve 1.4 g of Na2-EDTA in 100 ml bidistilled H2O.


• Fix 30 min in solution A.
• Wash 15 min in solution B.
• Wash 3 times 5 min with bidistilled H2O.
• Sensitize 2 min in solution C.
• Wash 3 times 30 sec with bidistilled H2O.
• Stain 25 min in solution D.
• Wash 3 times 1 min with bidistilled H2O.
• Develop 5 - 10 min in solution E (development solution).
• Stop 10 min in solution F.
• Wash with bidistilled H2O.

• Wear gloves and work under a laminar flow hood if possible to limit keratin contamination.
• Cut gel band/spot with a clean scalpel on a clean glass plate or Petri dish.
• Maximise protein concentration by cutting bands or spots where staining is the strongest.
• Transfer to Eppendorf tubes or 96-well PCR plates.
• Note: Total gel volume is important, as well as the ratio incubation buffer / gel volume. It is thus possible to digest one or more gel pieces, but the volumes of all washes and incubation steps have then to be adjusted accordingly.