Bio-Rad PROTEAN® i12™ IEF System - Manual (Page 44)

PROTEAN i12 IEF System Instruction Manual 40 | PROTEAN i12 IEF System Instruction Manual
To prevent protein contamination, for
example from skin keratin, wear laboratory
gloves when handling IPG strips and the
apparatus and solutions used in IPG strip
preparation.
Rehydration Solution
Prepare or dilute samples into a rehydration or
loading solution that contains urea, a nonionic
or zwitterionic detergent, carrier ampholytes, a
reducing agent such as dithiothreitol (DTT), and
bromophenol blue tracking dye (Table A.1). Optimum
composition depends on the sample, and the
guidelines in Table A.1 should serve as a starting
point for any optimization; additional or alternative
components may be useful as well. For more
comprehensive guidelines, see 2-D Electrophoresis
for Proteomics: a Methods and Product Manual
(bulletin 2651).
Appendix A
Reagent and Sample Preparation
Protein Sample Loads for IEF
The total amount of protein to load per IPG strip
depends on the sample, the pH range and length of
the IPG strip, and the detection system used (Table
A.2). Below are guidelines for protein loads that
produce acceptable 2-D patterns. In general:
■
Use less protein for silver staining and more for
Coomassie Blue staining. Fluorescent stains such
as Flamingo
™ , Oriole ™ , and SYPRO ® Ruby have a
wider dynamic range and a correspondingly wider
tolerance for protein load
■
Samples of greater complexity have protein mass
distributed among a larger number of protein
species, and narrow pH ranges have less sample
protein focusing within the pH range of the IPG
strip. Increased protein loads may, therefore, be
required for samples of higher protein complexity
and for narrow-range separations
■
The maximum that can be loaded onto each IPG
strip is 500 µg for 7 cm, 1 mg for 11 cm, 3 mg for
17 cm/18 cm, and 4 mg for 24 cm IPG strips
■
In some cases, overloading of protein is
acceptable and can help to reveal low-abundance
proteins of interest