- Acrylamide and bisacrylamide are neurotoxins when in solution. Avoid direct contact with the solutions and clean up spills.
- For casting multiple gels, use the Mini-PROTEAN® 3 multi-casting chamber, PROTEAN® II multi-gel casting chamber, or PROTEAN® Plus multi-casting chamber.
- Use only high-quality reagents, especially acrylamide monomers, to avoid polymerization problems.
- Proper degassing and filtering of the casting solution is critical for both reproducibility of the polymerization (oxygen removal) and the avoidance of problems related to mass spectrometry (keratin contamination).
- A temperature of 23–25°C is best for degassing and polymerization; equilibrate the stock solutions to room temperature.
- APS/TEMED-initiated reactions should proceed for at least 2 hr to ensure maximum reproducibility of pore size.
- Make fresh APS solution every day for best performance.
- Replace TEMED every three months because it is subject to oxidation, which causes the gradual loss of catalytic activity.
- The glass plates must be clean and free of chips. Clean glass plates with ethanol and lint-free cloths before use.
- The height of the stacking gel should be at least 2x the height of the sample in the well. This ensures band sharpness, even for diluted protein samples.
- Store gels flat in the fridge at 4°C. Do not freeze. Wrap handcast gels tightly in plastic wrap with combs still inserted.
- Run handcast gels with discontinuous buffer systems right after gel casting because the buffer discontinuity (pH and ionic strength) gradually disappear during storage. SDS-PAGE gels are not stable at pH 8.8 over a longer time period.
For more about acrylamide polymerization, refer to Bio-Rad bulletin 1156.
Premade Buffers and Reagents
Electrophoresis buffers and reagents are available as individual reagents or as premixed gel-casting, sample, and running buffers. Use of commercially prepared, premixed buffers helps save time but also helps to maximize reproducibility, avoid potential mistakes in buffer concentration and standardize electrophoresis runs, as they are made with electrophoresis-purity reagents and are quality controlled for reproducible results. There are no reagents to weigh or filter; just dilute with distilled or deionized water.
AnyGel stands provide stabilization and access to gels for casting and sample loading. The clamping mechanism secures gels cassettes vertically without excess pressure. They are available in two sizes, single- and six-row.
Multi-casting chambers are sued to cast multiple gels of various thicknesses simultaneously. Acrylic blocks act as space fillers when fewer than the maximum number gels are cst. These chambers work in concert with the gradient formers through a bottom filling port to ensure reproducibility. Multi-casting chambers are available for casting gels for the Mini-PROTEAN®, PROTEAN® II, and PROTEAN® Plus systems.
Gradient gels have a gradient of acrylamide concentration that increases from top to bottom. To create this gradient, the acrylamide solution must be mixed in a gradient former before being introduced into the gel cassette. Typically, two solutions are prepared: the light solution (equivalent to the lowest %T in the range to be poured) and a heavy solution (equivalent to the maximum %T to be poured). The most common gradient gel contains 4–20% acrylamide; however, the range of acrylamide concentrations should be chosen on the basis of the size of the proteins being separated.
Two gradient formers are available for PAGE systems. Depending on the gel format, prepare either a single gel using the gradient former, or couple the gradient former with a multi-casting chamber for the preparation of up to 12 gels simultaneously:
Multi-casting chambers and gradient formers.