Go to: content | top | bottom | search
 
 
You are hereUNIL > Center for Integrative Genomics > Protein Analysis Facility > Protocols

Protocols

MS-compatible Coomassie Blue R staining procedure (fast, simple) | Cell lysis protocol for 2D-PAGE | Colloidal Coomassie Brilliant Blue staining procedure | MS-compatible silver staining procedure | Gel cutting
 

MS-compatible Coomassie Blue R staining procedure (fast, simple)

  • 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.

TOP ^

Cell lysis protocol for 2D-PAGE

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

TOP ^

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).

    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.

TOP ^

MS-compatible silver staining procedure

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.

TOP ^

Gel cutting

  • 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.

TOP ^

Search:
 Go
 
rss/atom
Génopode - CH-1015 Lausanne  - Switzerland  -  Tel. +41 21 692 56 76  -  Fax +41 21 692 57 05
Swiss University