5 tips for good bands with SDS-PAGE protein gels
August 9th, 2017
The aim of this short post is to provide a few easy tips that can help with getting consistently good bands when running homemade and precast SDS-PAGE protein gels. If you run protein gels you may already know some of these tips, but we hope you will find some useful information regardless.
Consider heating and/or using reducing agents to prepare samples
While it is sometime overlooked, heating samples mixed will loading buffer can help fully denature all the proteins, which is important for consistently separating proteins only by molecular weight. On the other end of the spectrum, heating for too long or at too high a temperature can be detrimental as well. We find that heating samples for 10 Minutes at 70°C strikes the right balance for most samples.
Similarly, reducing samples before electrophoresis breaks possible disulphide bonds which would impair full denaturation of some proteins. The most commonly used reducing agents are beta-mercaptoethanol (BME) or dithiothreitol (DTT). Their efficiency depends on the pH of the sample buffer used.
Rinse and fill wells with water
When removing the comb of a gel (or when using a precast gel without a comb), we recommend rinsing and filling the wells with deionised water before loading the gel and filling the tank with running buffer. The density difference between the samples and water is greater than between the samples and running buffer. This means the samples will sink and settle to the bottom of the wells more quickly after loading. This also reduces the likelihood that the trailing ion from the running buffer (for example, Glycine, MES, MOPS or Tricine) will diffuse into the gel below the samples, which would disrupt stacking.
Load any empty wells with sample buffer
If any of the wells of a gel are not being used, the empty ones can be filled with 1X sample buffer by simply loading the same amount of buffer as total volume of a sample. This way each well has a similar ion content. This minimizes any edge effects on each lane of the gel.
Run straight away after loading samples
In most gel systems the sample buffer ions, their concentration, and their pH are similar to the gel. Even in traditional Laemmli gels, these conditions are similar to the stack. However, ions from the sample could diffuse into the gel and disrupt stacking. Once gel is in contact with running buffer in discontinuous systems, it is imperative to start running quickly as the run buffer ions should not be allowed to diffuse into the gel, which will disrupt stacking.
Keep gel temperature low and even
The local temperatures of the gel affect how samples migrate. High or uneven temperatures across the gel are often the main reason for smiling and other issues. There are many ways to keep the temperature low and even, but the easiest one is to fill the outer part of the tank with running buffer up to the bottom of the wells. This way heat dissipates almost homogeneously from the whole gel. This cools down the whole gel and avoids large local temperature differences.
If you have any question about the science behind these tips, or any other question about SDS-PAGE protein gels, please contact us, and our scientists will be in touch shortly. If you have any other suggestions for blog post topics, please let us know!