Bio-Rad ReadyPrep™ 2-D Starter Kit - Manual (Page 5)

Section 4
Instructions for Use
Introduction
This 2-D starter kit allows you to successfully separate a complex protein extract by 2-D PAGE
using IPG strips and the Bio-Rad PROTEAN IEF cell. The buffer formulations as well as the
following protocol have been optimized for the E. coli protein sample included in the kit. Before
beginning, decide which length IPG strips you wish to use and how many you wish to focus. The
kit contains sufficient reagents for the separation of the E. coli protein sample on six 17 cm IPGs,
ten 11 cm IPGs or sixteen 7 cm IPGs. The user has the flexibility to choose among three SDS-
PAGE formats (7 cm IPG strips and Mini-PROTEAN
® 3 Ready Gel precast gels, 11 cm IPG strips
and Criterion ™ gels or 17 cm IPG strips and PROTEAN ® II Ready Gel ® precast gels). The mini and
Criterion formats have the advantage that the entire 2-D electrophoresis process can be complet-
ed within a 24 hour period. The flow chart in Figure 1 can be used to estimate the amount of time
necessary to complete each of the steps of the 2-D process.
The instructions below describe the use of the E. coli protein sample with six IPG strips. Two of
these strips will be stained after the first dimension to illustrate the successful completion of the
IEF step. The remaining IPGs will be carried through the second-dimension separation. The SDS-
PAGE gels can be stained with either Bio-Rad’s Bio-Safe Coomassie stain or with Coomassie
Brilliant Blue R-250 stain.
3
The ReadyPrep 2-D starter kit has been optimized for pH 4-7 ReadyStrip IPG strips and either
Bio-Safe Coomassie or Coomassie Brilliant Blue R-250 stain. Strips of pH 5-8 can be used but
the results will not match the enclosed gel images. Strips of pH 3-10 are not recommended
unless the protein sample is further diluted into rehydration/sample buffer at least 4-fold. Other
stains can also be used, but again the protein sample needs to be diluted if a more sensitive
stain is used.