Gel Loading Dyes
The gel loading dye helps to increase the density of your samples so they stay in the well. A tracer dye is useful to visualize filled wells and gives an estimate of progression of electrophoresis. Finally, for SDS-PAGE gels, the SDS present in the loading dye helps to denature proteins.
1. Agarose Loading Buffer (6x)
Section titled “1. Agarose Loading Buffer (6x)”Common dyes and how they approximate linear DNA on an agarose gel. These are very rough estimates.
| Dye | 0.5-1.5% agarose | 2.0-3.0% agarose | CAS number | Sigma Catalog # |
|---|---|---|---|---|
| Xylene cyanol | 10-4 kbp | 750-200 bp | 2650-17-1 | Sigma X4126 |
| Cresol Red | 2-1 kbp | 200-125 bp | 62625-29-0 | Sigma 114480 |
| Bromophenol blue | 500-400 bp | 150-50 bp | Sigma B8026 | |
| Orange G | <100 bp | ? | Sigma O3756 |
The precise amount of dye is not important. However, it is advisable that you prevent overlay of dye and expected DNA size. For example, if you are expecting a band of 200-400nt, bromophenol blue will obscure your product a bit.
1.1 Recipe
Section titled “1.1 Recipe”Sucrose & xylene cyanol / bromophenol blue (6x) Combine:
| Component | Amount | Final concentration |
|---|---|---|
| sucrose | 4g | |
| bromophenol blue | ~20mg | |
| xylene cyanol | ~20mg | 0.25% |
| dH2O | to 10mL |
Storage: Store at 4°C to avoid mould growing in the sucrose. 10mL of loading buffer will last for years.
Recipes borrowed from OpenWetWare.
2. SDS-PAGE Loading Buffer (4x)
Section titled “2. SDS-PAGE Loading Buffer (4x)”For preparation and loading of protein samples onto a gel for SDS-PAGE analysis (Western blot/protein blot). SDS contained in the sample buffer makes proteins negatively charged proportionally to their length. 2-mercapto-ethanol/DTT breaks disulphide bonds. Glycerol makes the sample buffer more dense than the surrounding running buffer of the protein gel, enabling easy loading into the gel pockets.
2.1 Recipe
Section titled “2.1 Recipe”To make 10 mL of 4x stock, combine:
| Component | Amount | Final concentration (1x) |
|---|---|---|
| 1M Tris-HCl pH 6.8 | 2.0 ml | 50 mM Tris-HCl pH 6.8 |
| SDS | 0.8 g | 2% SDS |
| 100% glycerol | 4.0 ml | 10% glycerol |
| 14.7 M β-mercaptoethanol | 0.4 ml | 1% β-mercaptoethanol |
| 0.5 M EDTA | 1.0 ml | 12.5 mM EDTA |
| bromophenol Blue | 8 mg | 0.02 % bromophenol blue |
Storage: Store in aliquots at -20oC.
Use of loading buffer: Dilute protein sample 1:3 into 4X sample loading buffer. Load on acrylimide gel in SDS-PAGE buffer.
Recipes borrowed from OpenWetWare.