When preparing samples for reducing gel electrophoresis, any of the following reducing agents may be used:
- NuPAGE® Reducing Agent
- Dithiothreitol (DTT), 50 mM final concentration
- ϐ-mercaptoethanol, 2.5% final concentration
- Tris(2-carboxyethyl)phosphine (TCEP), 50 mM final concentration
We recommend only adding the reducing agent to the sample up to an hour before loading the gel. Avoid storing reduced samples for long periods, even if they are frozen. Reoxidation of samples occurs during storage and produces inconsistent results. For optimal results, we do not recommend running reduced and nonreduced samples on the same gel. If they must be applied to the same gel, do not run reduced and nonreduced samples in adjacent lanes; the reducing agent from the reduced samples may affect the nonreduced samples if they are in close proximity.
Heating the sample at 100°C in SDS-containing buffer results in proteolysis (Anal Biochem 225:351 (1995)). We recommend heating samples for denaturing electrophoresis (reduced or nonreduced) at 85°C for 2–5 minutes for optimal results. Do not heat the samples for nondenaturing (native) electrophoresis or zymogram gels.
High Salt Concentrations in Samples
High salt concentrations result in increased conductivity that affects protein migration, and can result in gel artifacts in adjacent lanes containing samples with normal salt concentrations. Perform dialysis, or precipitate and resuspend samples in lower-salt buffer prior to electrophoresis.
Guanidine-HCl in Samples
Samples solubilized in guanidine-HCl have high ionic strength and produce increased conductivity similar to the effects of high salt concentrations. In addition, guanidine precipitates in the presence of SDS, leading to various types of gel artifacts. If possible, change the solubilization agent by dialysis prior to electrophoresis.
Consider the following when performing electrophoresis of cell lysates:
- Genomic DNA in the cell lysate may cause the sample to become viscous and affect protein migration
patterns and resolution. Shear genomic DNA to reduce viscosity before loading the sample.
- Cells lysates contain soluble and insoluble fractions. The size of each fraction depends on the type of
sample being analyzed. The nature of the insoluble fraction may result in altered protein migration
patterns and resolution. Separate the two fractions by centrifugation and load them on separate lanes
- If radioimmunoprecipitation assay (RIPA) buffer is used in cell lysis, subsequent blotting of proteins less
than 40 kDa may be inhibited due to the presence of Triton® X-100 in the buffer.