4 sample preparation, 5 running conditions, 3 sds-page buffers – Bio-Rad Mini-PROTEAN® TGX™ Precast Gels for 2-D Electrophoresis User Manual
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3.3 SDS-PAGE Buffers
Running buffer (1x)
25 mM Tris, 192 mM glycine, 0.1% SDS
Dilute 100 ml 10x stock (catalog #161-0732) with 900 ml deionized water (diH
2
O).
Sample buffer (2x)
62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% (v/v) glycerol, 0.01% bromophenol
blue, 5%
β-mercaptoethanol or 100 mM DTT (added fresh)
Use Laemmli sample buffer (catalog #161-0737) and add
β-mercaptoethanol or
DTT before use.
Sample buffer (4x) 250 mM Tris-HCl, pH 6.8, 4% LDS, 40% (w/v) glycerol, 0.02% bromophenol
blue, 15% beta-mercaptoethanol or 200 mM DTT (added fresh)
Use 4x Laemmli sample buffer (catalog #161-0747) and add
β-mercaptoethanol
or DTT before use.
3.4 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 sample buffer with added reducing agent.
2x: dilute 1 part sample with 1 part sample buffer.
4x: dilute 3 parts sample with 1 part sample buffer.
For nonreducing conditions, omit the reducing agent.
3. Heat the diluted sample at 90–95°C for 5 min or at 70°C for 10 min.
3.5 Running Conditions
Run conditions and times are approximate. Run times represent the time required for the dye front
to reach the line at the bottom of the cassette. Conditions may vary depending on water and buffer
conductivity, which vary from one lab setting to the next. Multiply current by the number of gels run.
Table 3.1. Standard running conditions for SDS-PAGE in the Mini-PROTEAN Tetra cell.
Gel
Optimum Range
Run Conditions
Run Time
7.5%
40–200 kD
10%
30–150 kD
300 V constant:
12%
20–120 kD
Starting current (per gel): 55–75 mA 15–20 min
4–15%
20–250 kD
Final current (per gel): 45–70 mA
(Fill outer buffer volume
4–20%
10–200 kD
to the 4-gel mark)
Any
kD
10–100
kD
See Appendix B for buffer formulations. Do not adjust pH.
Mini-PROTEAN Precast Gels