EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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Semi-Colloidal Coomassie (at 40/60 °C)

This staining takes a little longer than the normal “Hot Coomassie Staining”.
The advantages is:
   a) More sensitivity
   b) Also peptides are visible
   c) Also lipophylic proteins stay in the gel during staining
   d) Ideal for preparative 2D-stainings

The semi colloidal Coomassie-staining is staining and fixing at the same time.
The Phosphoric acid is less lipophilic than the Acetic acid.
This results in a slightly colloidal staining.
The 3% Acetic Acid is only used for  getting the Coomassie better solubilized and we achieve therefor a „tank-clean“ staining recipe.
We recommend this method for peptides and preparative 2D-gels.

Stock solutions: (Volumes are given for normal sized gels, 12 × 26 cm)
staining solution:  0.01 % (w/v) Coomassie R 350 (GE: 1 tablet) in 2.8 L H2Odist
                      
+ 0.5% (w/v) Phosphoric acid (18 mL) + 3% HAc  (90 ml HAc) —> fill up to 3 L
destaining solutiont:  200 mL 10% HAc; 400 mL for large gels.
preserving solution: 200 mL 10% (v/v) glycerol; 400 mL for large gels

Mai 30, 2018

The “Semi-colloidal Hot Coomassie Staining
 in a “Steel Tray”:

The staining tray is placed on a magnetic heater.
The gel lays gel face-down on the grid.
Below the grid stirs the magnetic bar.

Important:
Because our NF-films do not stay alcoholic liquids with more than 30% and no hot staining methods
 all our proteomic gels are available also on normal film support.
These products are coded with this yellow colour in the background.
If NF-film supported gels should be Coomassie-stained after fluorescence-visualisation (spot-picker!)
we suggest the “Semi-colloidal Coomassie” at 50 °Celsius

2D-gels:
If Pharmalytes were used in the first dimension this 2 washing steps should be done first:

Washing programm:                     40% EtOH/10% HAc for 30 min, 20% EtOH/10% HAc for 30 min

Staining Programm

solution

standart gel-matrix

2DGel Matrix

temp (°Cels.)

fresh staining solution
(see picture “Hot Coom.Stain”, fume cupboard!)

1-2 h
(or overnight)

2h
(or overnight) 

65 (normal film)
 50 (NF-film)

destaining solution in a tray
(rocking platform)

2 x 20 min
(or overnight)

2 x 30 min
(or overnight)

ambient

preserving solution (in a tray)

30 min

30 min

ambient

GE Peptide-marker and bacterium-lysate on 1DGel 15% 25S, stained with 65 degrees Celsius
K.Buettner, Greifswald

PeptidemikrogrammKlein

1 mg E.coli extract (Sigma) on a vertical gel 12.5%T casted on a non-fluorescent film. Stained at 50 degrees Celsius

pre-flood#3Klein