ThermoFisher Scientific E-PAGE Gels, Техническое руководство

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12
Guidelines for Sample Preparation,
Continued
Method 2: Lumio ™
Detection
A brief protocol to prepare samples for specific detection of Lumio ™ fusion
proteins using the Lumio ™ Green Detection Kit is described below.
For details on the Lumio
™
Green Detection Kit, refer to the manual available at
www.lifetechnologies.com or contact Technical Support (page 72).
1. Refer to the Lumio

Detection manual for details on each type of protein.
Prepare protein samples as follows:
Protein Sample Sample
Volume
Lumio ™ Gel Sample Buffer (4X)
Volume
Bacterial samples 7.5 µL 2.5 µL
Mammalian lysate 7.5 µL 2.5 µL
Partially purified
sample
7.5 µL 2.5 µL
Purified sample 7.5 µL 2.5 µL
In vitro expressed 10 µL Not needed*
*There is no need to add Lumio
™
Gel Sample Buffer (4X), as the sample is
already prepared in this buffer.
2. Thaw the Lumio
™
Green Detection Reagent and mix well.
3. Add 0.1 µL Lumio ™ Green Detection Reagent to the protein samples from
Step 1 in a fume hood. Mix well. Return the Lumio ™ Green Detection
Reagent to –20
°
C immediately after use.
4. Incubate the samples at 70
°
C for 10 minutes.
5. Allow samples to cool for 1–2 minutes and centrifuge briefly at maximum
speed in a microcentrifuge.
6. Thaw the Lumio ™ In-Gel Detection Enhancer and mix well. Add 1 µL of
Lumio ™ In-Gel Detection Enhancer to the samples.
7. Mix well and incubate the samples at room temperature for 5 minutes.
Return the Lumio
™ In-Gel Detection Enhancer to –20
°
C immediately after
use.
8. Proceed to Loading E-PAGE

Gels, page 13.
When performing electrophoresis of Lumio
™
fusion proteins, we recommend
extending the run time of the gel for an additional 2 minutes to prevent the
formation of a fluorescent dye front.