EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

Datenschutzerklärung >>

Blakesley IEF-Coomassie Stain / Roti-Blue (Roth)

ANALYTICAL BIOCHEMISTRY 82, 580-582 (1977)

Stain preparation.
Coomassie brilliant blue G250 (xylene brilliant cyanin G) was purchased from K and K Laboratories, Inc. (Catalog No. 27551).

To a 0.2% (w/v) aqueous solution of the dye was added an equal volume
of 2 N H2SO4.
After the solution was mixed well, it was allowed to stand for at least 3 hr. The resulting precipitate was removed by gravity filtration through Whatman No.1 paper.
The volume of the clear brown filtrate was measured and one-ninth volume of 10 N KOH was added, producing a dark purple solution.
To the dark purple solution, 100% (w/v) trichloroacetic acid was added to a final concentration of 12% (w/v).

The resulting clear light blue solution was ready for use.
The staining solution can be stored for several months without loss of
effectiveness. It may be reused several times, but the pH of the solution
must be maintained below 1.0.
Staining procedure.
After electrophoresis, the polyacrylamide gel was added directly to the staining solution. For example, a 0.7 x 10 cm
cylindrical gel was placed in a test tube with 10-15 ml of stain.
Within 30 min, bands containing 5-10 ug of protein were evident. Color development for maximum sensitivity was achieved in 5-8 hr.

Gels could be stored in the staining solution without having the gel backgrounds stain significantly. If the gels were stored in water (10 -15 ml), there was a marked color intensification of the stained protein bands and gel background reduction producing optimal sensitivity. However, if the gels were placed in solutions containing sulfuric acid, trichloroacetic acid, or acetic acid, there was a rapid loss of intensity of the stained protein bands

Ready-To-Use solution from Roth (Karlsruhe): Roti-Blue
Roti-Blue (#A152.1) is a 5 X concentrated solution for fixing and staining (SDS)
Sensitivity is 3-5 fold higher than other Coomassie-stainings

EDC’s recipe and procedure for urea-containing IEF-gels

Mai 30, 2018

bidirekt-CoomViol#2ASKlein

Coomassie-violet in urea-containing gels

MaisASKlein

Blakesley: factor ~3-4 more sensitive in urea-cont. gels

fixing

100 ml H2O

+ 12.5 g TCA

30 min

washing

70 ml H2O + 30 ml Methanol

+ 1 ml Phosphoric Acid

15 min

staining on shaker

mix: 60 ml H2O + 20 ml Methanol

stir in: 20 ml Roti Blue*

3 h - overnight

washing

75 ml H2O + 25 ml Methanol

---

5 min

preserving for drying

3% (v/v) Glycerol

---

20 min

* shake the bottle well!

Maize-prolamins by R.Knoblauch (LUFA Karlsruhe)

large sized gels: double volumes

IEFCerealKlein