EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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IEF-Coomassie Stain (methanolic and ethanolic)

Mai 30, 2018

Do not use these two staining recepis when lipophilic proteins should be visualized. A non-alcoholic staining should be used ---> Coomassie-Violet Staining. Glyco-peptides ---> Blakesley

1. Staining with Coomassie Blue G-250 (MeOH)
Stock solutions:

TCA:  20% TCA: Dilute 40 ml of 100% TCA (w/v) to 200 ml
A:       0.2 % CuSO4 / 20 %  acetic acid (2 g of CuSO4 in 1L of 20% HAc)
B:       0.04 % Coomassie G-250 in 60 % methanol (0.4 g Coomassie G-250 in 1L 60 % methanol)
C:       50% (v/v) methanol

Staining programme:
Fix:            15 min in 200 ml 20 % TCA (at room temperature)
Wash:         2×1 min in 200 ml wash solution (mix equal amounts of A and C)
Stain:         45 min in 200 ml of staining solution (mix equal amounts of A and B)
                 (Heat the staining solution to 50 °C while stirring, see figure 8)
Wash:          2×5 min in 200 ml wash solution (mix equal amounts of A and C)
Destain:       2-3 × 15 min in wash solution* (in a tray), A and C
Impregnate:  5 min in 200 ml 5 % (v/v) glycerol
Dry:            air-dry (leave at room temperature)
                        * = visual control


IEF-Coomassie Stain (ethanolic)

New recipe: we replaced the methanol with ethanol!
2. Staining with Coomassie Blue G-250 (EtOH)
Stock solutions:

TCA:
  20% TCA: Dilute 40 ml of 100 % TCA (1kg/l) to 200 ml
A:      0.2% CuSO4 / 20%  acetic acid: Dissolve 2 g of CuSO4 x 5 H2O in 1 l of 20% (v/v) acetic acid.
B:     0.04% Coomassie G-250 in 25% (v/v) ethanol: 0.4g Coomassie G-250 + 250 ml Ethanol + 750 ml H2O
C:      20% (v/v) Ethanol: 200 ml Ethanol + 800 ml H2O

Staining programme:

Fix:              20 min in 200 ml 20% (w/v) TCA
Rinse            5 min 200 ml H20 Bidest
Wash:          5 min in 200 ml wash solution (mix equal amounts of A and C)
Stain:           30 min in 200 ml of staining solution (mix equal amounts of A and B)
                  (pre-heat the staining solution to 50°C while stirring). See the picture below!
Destain:        2-3 x 20 min* in wash solution (in a tray), best is 1 x overnight
Impregnate:   5 min in 200 ml 5 % (v/v) glycerol
Dry:              leave at room temperature, overnight
                          * = visual control

The assembling of the Hot Coomassie Staining:
A steel tray is placed on a magnetic heater.
The gel lays gel face-down on the grid.
Below the grid the magnetic bar stirs