Bio-Rad PROTEAN® i12™ IEF System - Manual (Page 45)

PROTEAN i12 IEF System Instruction Manual
Appendix A Sample and Reagent Preparation
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Table A.2. Rehydration volumes and sample loads. Protein concentration in samples prepared for IEF can be difficult to
determine accurately due to interference from detergents, reductants, and other sample components. For best results in protein
quantitation, use the RC DC
™ protein assay kit (catalog #500-0121 and #500-0122).
IPG Strip Length
7 cm 11 cm 17 cm 18 cm 24 cm
Rehydration Solution 125 μl 200 μl 300 μl 315 μl 450 μl
Protein Load
Coomassie (Brilliant) Blue 50–100 µg
100–200 µg 200–400 µg 200–400 µg 400–800 µg
Fluorescent stains 5–100 µg 20–200 µg 50–400 µg 50–400 µg 80–800 µg
Silver stains 5–20 µg 20–50 µg 50–80 µg 50–80 µg 80–150 µg
Table A.1. IPG strip rehydration solution composition. Vary
the concentrations of the individual components as needed
within the range given.
Concentration
Component Standard
Range
Urea 8 M 7–9.5 M
Thiourea — 0–2 M*
CHAPS 2% 1–4%
DTT 50 mM 15–100 mM
Ampholytes (w/v)** 0.2% 0.1–0.4%
Bromophenol blue 0.001% 0.001%
* Thiourea may be used with urea for more effective solubilization
and focusing of hydrophobic proteins (Rabilloud et al. 1997).
**For example, Bio-Lyte ampholytes. Use the pH range
corresponding to the IPG strip selected.