Bio-Rad Mini-PROTEAN® TGX™ Precast Gels for 2-D Electrophoresis - Manual (Page 23)

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Running buffer (1x) 100 mM Tris, 100 mM Tricine, 0.1% SDS
Dilute 100 ml 10x stock (catalog #161-0744) with 900 ml diH
2
O
Sample buffer 200 mM Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 0.04% Coomassie
(catalog #161-0739) Brilliant Blue G-250, 2% β-mercaptoethanol or 100 mM DTT (added fresh)
6.4 Sample Preparation
1. Determine the appropriate concentration of sample to load (depends on the load volume and the
detection method used; see Chapter 10 for approximate stain sensitivities).
2. Dilute the sample with at least an equivalent volume of sample buffer (catalog #161-0739) and
reducing agent ( β-mercaptoethanol, for example). Heat the diluted sample at 90–95°C for 5 min,
or at 70°C for 10 min.
For example, combine: 5 μl sample
4.75 μl Tricine sample buffer (catalog #161-0739)
0.25 μl β-mercaptoethanol (catalog #161-0710)
10 μl total volume
6.5 Running Conditions
Table 6.1. Running conditions for one (1) Mini-PROTEAN Tricine gel in the Mini-PROTEAN Tetra cell . Run conditions
and times are approximate and assume a constant voltage of 100 V. When running more than one gel, current will differ.
16 .5% Gels 10–20% Gels
Power conditions 100 V constant 100 V constant
Expected current (per gel)
Initial 65 mA 65 mA
Final 35 mA 35 mA
Run time 100 min 100 min
See Appendix B for buffer formulations. Do not adjust pH unless instructed to do so.
6.3 Peptide Analysis Buffers
Instruction Manual and Application Guide