EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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Checking the quality of the first dimension

To obtain good results it is important to ensure that the first dimension (IPG-strip) has run properly.
Before the equilibration steps and the start of the second dimension it would be very important to know if:

     1.   Did the first dimension run properly?
     2.   Did the sample enter the strip?

1.
A simple way is to view the IPG-strip using an oblique light source.
In a properly run first dimension the high concentrated Ampholyte-components will separate into sharp bands causing shrinkage of the strip at that point which will appear as ‘valleys’ when examining the surface. The pH-gradient worked well if you could see this “ washboard”.

Mai 30, 2018

IPG-BandsAS

With a little practise, the main bands of the Ampholyte-components can be visible at the strips surface:
a lamp should be mirrored at the strips surface.

2a.
Using different amphoteric colours in the strip and in the sample the entrance of the sample in the strip could be controlled.

Ladder  LM:  green – 2.0; red – 3.3; orange 4.3; lavender- 5.3; red – 6.2; red 7.1; strawberry – 8.0;  maroon (brown-red)  – 9.0; yellow - 10.1

Ladder LN
: purple -  2.6; orange – 3.9; purple -  4.7; yellow - orange – 5.7; lavender – 6.8;
red – 7.5; brown-red  - 8.6; blue  -  9.5; strawberry – 10.3

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IPG-strip pH 4-7, 24 cm
(lilac colour from the sample...
 amphoteric colour mix “LM” (lilac & red)

acidic amphoteric colours in the sample

Use of different amphoteric colours in the sample and in the strips ...

IPG-strip pH 7-11, 11 cm
(amphoteric colour mix “LN” from the strip
+ lilac and red from the sample)

basic amphoteric colous in the strips

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Animation by: Doc. RNDr. Karel Slais

amphoteric colours

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Testing the optimal sample application point.
Lilac and red colour at the anode come from the sample, the other colours from the strip!

2b.
Using prelabelled fluorescent proteins opens the possibility to scan the ready focused IPG-strip in a scanner before equilibrating and applying it on the second dimension.

Test IPG Strips [Cy5]

Scanning the IPG-strips after focusing - to verify the fluorescent bands after the 1st dimension.....