EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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IEF-Electrophoresis Trouble Shooting

Mai 30, 2018

General mistakes
When mounting up a horizontal electrophoresis run, several mistakes can be done.
Normally their occur already after starting by watching the power supply and the migration of the dyes:

No currant, 0 mA, no migration of the dyes:
1   The cables inside the chamber are not switched properly
2   The platin electrodes are not in contact with the electrode strips
3   The electrode strips are not in contact with the gel (no overlapping zone)
4   The power supply is not programmed in a correct way (no volt = no mA)

 Too fast migration of the dyes, gel is sweating and later burning:
The dyes of the samples and the gel should migrate around 0.5 mm per minute, not more!
1   Too much currant, too high mA value:
     a   Power supply is not programmed correctly
     b   A part of the gel runs with the conditions of a whole gel
2   The cooling device is not running correctly, or it is not pumping really through the cooling plate

Samples
The sample concentration should not be too high!
As a sufficient pre-test the protein test-stick “Albym-Test” (Boehringer) can be used; available in every pharmacy (for urinary proteins).

Protein Test Stick

Protein Concentration

Stick Colour

Sample-Dilution (Coomassie)

negative

 

no dilution

0.3 g/l

 

no dilution

1.0 g/l

 

10ul + 30ul

5.0 g/l

 

10ul + 90ul

Even in a excellent performing electrophoretic system you will not get automatically acceptable results from every sample! There will be always 'trouble-making-samples', appearances, reasons and remedies see below:

[Sample Spread]
[Curved Bands]
[Sample Salt]
[Lipoproteins]
[Glyco-peptides]
[Contact-Problems]
[Blue Lines]

1. Samples spreading over the gel

2. Bands occur curved

3. Lanes are spreaded lateral

4. No proteins visible after staining

5. No peptides visible after staining

6. Separation partially curved

7. Iso-pH lines visible after staining