Bio-Rad ReadyPrep Protein Extraction Kit (Soluble/Insoluble) - Manual (Page 11)

7. Carefully remove and pool the supernatant with the
supernatant previously collected. The combined
supernatants are referred to as the Soluble fraction.
8. Before performing the extraction of the pellet,
complete the preparation of the 2-D
Rehydration/Sample Buffer 1. Prepare only enough
complete 2-D rehydration/sample buffer 1 for the
number of extractions being performed. Each
extraction will require 0.5 to 1.0 ml of the solution.
The solution is prepared by adding 10 µl of ReadyPrep
TBP reducing agent and the appropriate ampholyte to
0.2 % (w/v) to every 1 ml of reconstituted 2-D
Rehydration/Sample Buffer 1. The ampholyte is
chosen to match the pH range of the IPG strip to be
used for 2-D analysis, though for most applications,
Bio-Rad’s Bio-Lyte 3/10 ampholyte (catalog # 163-
2094) can be used. If desired, other additions such as
protease inhibitors can also be made at this time.
9. To the pellet, add 0.5 to 1.0 ml of complete 2-D
rehydration/sample buffer 1. Vortex or sonicate the
sample until the protein pellet is completely solubilized.
Note: If using sonication, care must be exercised to prevent
heating of the sample. The temperature of the sample
should not be allowed to rise above 30°C. Similarly, if the
sample becomes too cold, precipitation of the urea and
thiourea can occur. If this happens, gently warm the sample
until the precipitate dissolves before proceeding further.
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