Bio-Rad ReadyPrep™ 2-D Starter Kit - Manual (Page 7)

4.1 Sample Preparation
1 Remove the bottle of rehydration/sample buffer, the bottle of nanopure water and the vial
of E. coli protein sample from the kit. All 3 bottles have a GREEN cap.
2 Remove the desired number of pH 4-7 ReadyStrip IPG strips from the -20ºC freezer and
set aside. (Six strips are recommended: 2 to stain after IEF, 4 to be used for second-
dimension SDS-PAGE).
3 Place one disposable rehydration/equilibration tray of the same size as the IPG strips to
be run onto the bench with the sloped end facing to the right (See Figure 2).
4 Remove the crimp and stopper from the bottle of rehydration/sample buffer and reconsti-
tute the lyophilized powder by adding 6.1 ml of the nanopure water supplied with the kit.
Swirl the bottle gently at intervals until all the solids are completely dissolved. The bottle will
chill as the urea in the solids dissolves. The bottle can be warmed slightly in the palm of the
hand to expedite this buffer reconstitution process. Take care not to warm the solution above
30ºC.
5 Open the vial of E. coli protein sample and reconstitute the lyophilized protein by adding
2 ml of the freshly prepared rehydration/sample buffer. Replace the rubber stopper and
gently invert the vial 4 to 6 times to completely reconstitute the E. coli proteins. Do NOT place
the vial on ice as the urea will crystallize out of the solution.
4.2 Sample Application During Rehydration
The amount of reconstituted E. coli protein sample to load per IPG strip is indicated in
Table 1. These quantities are sufficient to easily visualize a large array of protein spots
when the second-dimension SDS-PAGE gel is stained with Bio-Safe Coomassie stain or
Coomassie Brilliant Blue R-250 stain.
1. Using Table 1 below, pipet the indicated volume of the reconstituted E. coli protein sample
as a line along the back edge of channel #1. The line of sample should extend along the
whole length of the channel except for about 1 cm at each end. Take care not to intro-
duce any bubbles which may interfere with the even distribution of sample in the strip
(See Figure 2. Sample loading of rehydration/equilibration trays).
Strip Length 7 cm 11 cm 17 cm
Sample Volume 125 µl 185 µl 300 µl
Protein Loaded 169 µg 250 µg 405 µg
Table 1.
The increased sensitivity of the Bio-Safe Coomassie stain over the R-250 stain allows you to
dilute the stock of E. coli protein sample up to 4-fold into rehydration/sample buffer and still
achieve excellent images. If you choose to stain the SDS-PAGE gels with silver stain (such as
Bio-Rad’s Silver Stain Plus stain, catalog # 161-0449) or a fluorescent stain (such as
SYPRO* Ruby Stain, catalog # 170-3125) use a 4-fold diluted E. coli protein sample (diluted into
rehydration/sample buffer) and the rehydration volumes per strip indicated below.
*SYPRO is a trademark of Molecular Probes, Inc.
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