EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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Iso-pH lines visible after staining

Mai 30, 2018

1. Cause in normal IEF CBB staining:
During the fixing process with 20% TCA the Ampholytes who are producing the pH-gradient should leave the gel completely. If certain Ampholytes will not do this then they get stained like the proteins because they are also Amino-carbon-acids.
Remedy:
If alcohol-containing staining recipes are used, the staining and the destaining solution should be more lipophylic.
Slightly increase the alcohol compound of the washing solution. See table below.
The increasing should be done very carefully because lipophylic proteins get soluble too and will be not visible anymore after the visualization!
If Serva-Violet staining is used no other Ampholytes should be inside the gel.

a. MeOH-based recipes: increase slightly the MeOH from 30% to 33%

b. ETC's EtOH-based recipe: increase slightly the EtOH concentration in the staining bath from 12.5% to 13%, in the destaining bath from 10% to 11%

IEFTS-stripes
Pfeil

Iso-pH lines visible

IEFTS-stripes2

alcohol concentration slightly increased

2. Cause in Serva-Violet staining: The IEF-gel must not contain other Ampholytes than Servalytes. Use ETC's FocusGel 2-6.

IEFTS-stripes3
Pfeil

IEF-gel contains not only Servalytes

IEFTS-stripes4

...only Servalytes