Bio-Rad ReadyPrep™ Protein Extraction Kit (Membrane II) - Manual (Page 13)

11. 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 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 2D analysis, though for most appli-
cations, 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.
12. Resuspend the membrane-protein pellet(s) from each
extraction with a total of 1.0–2.0 ml of complete 2-D
rehydration/sample buffer 1. Alternatively, the pellets
can be stored at –80°C for later analysis.
13. Vortex and/or sonicate the sample to completely
solubilize the proteins in the pellet.
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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