Bio-Rad ReadyPrep™ Reduction-Alkylation Kit - Manual (Page 9)

5. Add 15 µl of 0.5 M iodoacetamide for a final
concentration of 15 mM. Mix well by vortexing.
Incubate at room temperature for 1 hr.
Note: Longer incubation times may cause spurious
reactions between the iodoacetamide and the protein that
could create artifacts in mass spectrometry analysis.
6. Add an additional 12.5 µl of TBP to quench any
unreacted iodoacetamide. Mix well by vortexing.
Incubate at room temperature for 15 min.
7. Centrifuge at 12,000–16,000 x g for 5 min at room
temperature to pellet any insoluble material.
8. Dilute the protein sample to the appropriate final
volume in rehydration/sample buffer and load onto the
IPG strip. Refer to Section 5.1 to determine the
volumes of rehydration/sample buffer and amounts of
protein needed for different strip lengths and staining
methods. For further improvement of spot resolution,
especially for basic strips (pH 7–10), cup loading is
required. For information on cup loading, see the Cup
Loading Tray instruction manual (4006216) and bulletin
2812.
6
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