Performing Electrophoresis

General Protocol: SDS-PAGE

The following is a generalized protocol for running a Mini-PROTEAN^® TGX™ gel in the Mini-PROTEAN Tetra cell.

		Prepare buffers. a. Running buffer (1x): Dilute 100 ml of 10x stock with 900 ml diH2O. b. Sample buffer: Use Laemmli sample buffer.
		Prepare gels and assemble the electrophoresis cell. a. Remove the comb and tape from the gels and assemble the electrophoresis cell. b. Fill the inner and outer buffer chambers with running buffer. Fill the inner (upper) buffer chamber of each core with 200 ml of 1x running buffer. Fill the outer (lower) buffer chamber to the indicator mark for 2 gels (550 ml) or 4 gels (800 ml) with 1x of running buffer. At runs >200 V, fill the outer buffer chamber to the 4-gel (800 ml) mark.
		Prepare samples as indicated in the table below. Component Reducing Nonreducing   Sample 5 µl 5 µl Laemmli sample buffer 4.75 µl 5 µl β-mercaptoethanol 0.25 µl —   Total volume 10 µl 10 µl
		Heat samples at 90–100°C for 5 min (or at 70°C for 10 min). Load the appropriate volume of your protein sample on the gel.
		Connect the electrophoresis cell to the power supply and perform electrophoresis according to the following conditions:   Run conditions: 200 V   Run time: 31–39 min   Expected current (per gel): Initial 35–50 mA   Final 20–31 mA
		After electrophoresis is complete, turn the power supply off and disconnect the electrical leads. Pop open the gel cassettes and remove the gel by floating it off the plate into water.
		Stain and image the gel, using one of the protocols.

General Tips for Protein Electrophoresis

• When preparing running buffers, make the solution as written and do not titrate to a pH. The ion balance is set by the concentration of reagents; adjusting the pH alters this balance and leads to undesirable results
• Do not reuse running buffers
• Use 5–10 V per cm gel length for about 10 min during sample entry (or until the sample has concentrated at the starting point of the separation gel). Then continue with the voltage setting recommended in the instruction manual for the electrophoresis system you are using
• Use the voltage setting recommended in the instruction manual for the electrophoresis system you are using; excessive voltage leads to decreased band resolution, band smiling, and lane distortions
• When running multiple cells, use the same voltage for multiple cells as you would for one cell. Be aware that the current drawn will double with two cells (compared to one). Use a power supply that can accommodate this additional current and set the current limit high enough to permit this additional function
• To maximize reproducibility, maintain the temperature of the electrophoresis buffer at ~20°C with the help of a recirculating cooler

General Tips for Sample Loading

• The total protein amount loaded per lane depends on sample complexity and the sensitivity of the staining technique. Using 15–20 µg protein per lane for mini- or midi-format gels is a good starting point for complex protein samples when staining with Bio-Safe™ Coomassie stain. Determine the optimum protein load by running a dilution series of the sample
• For best resolution, load a concentrated sample rather than a diluted amount
• Centrifuge the sample solution for 10–15 min at >12,000 x g at 20°C before loading to remove insoluble material that may clog the pores of the acrylamide gel
• To avoid edge effects, add 1x sample buffer to the unused wells and never overfill wells
• Load samples either before or after placing the electrophoresis modules into the tank. Both methods produce acceptable results. In both cases, fill both the assembly (inner chamber) and the tank (outer chamber) with buffer
• Add running buffer to the cathode buffer reservoir first and then apply the sample on the stacking gel under the electrode buffer. Sample buffer must contain glycerol to stabilize the sample application zone in the sample well of the gel
• Use pipet tips designed for protein sample loading for best results. For example, Bio-Rad's Prot/Elec tips fit easily between vertical slab gel plates of 0.75 mm while maintaining a large bore for fast flow of sample
• Load the samples into the wells with a Hamilton syringe or a pipet with gel loading tips
• Load samples slowly to allow them to settle evenly on the bottom of the well. Be careful not to puncture the bottom of the well with the syringe needle or pipet tip
• If using Bio-Rad's patented sample loading guide, place it between the two gels in the electrode assembly. Sample loading guides are available for 9, 10, 12, and 15-well formats. Use the sample loading guide to locate the sample wells. Insert the Hamilton syringe or pipet tip into the slots of the guide and fill the corresponding wells

Troubleshooting

Evaluation of Separation