Cell lysis protocol for 2D-PAGE
Preparation of cell lysate for 2D PAGE
Cell Wash buffer
- 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
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
Colloidal Coomassie Brilliant Blue staining procedure
More sensitive than regular Coomassie, but also longer to do.
According to Candiano et al. (Electrophoresis 25, 1327–1333, 2004).
* orthophosphoric acid
* 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.
* 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.
MS-compatible silver staining procedure
Adapted from Blum et al., Electrophoresis
8, 93-99, 1987
- 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.