Tips for Optimal SDS-PAGE Separation

 

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate mixtures of proteins of variable complexity and allows you to determine sample protein composition, purity, whether you have a target of interest in your sample, and even some of its structural characteristics. Gel electrophoresis is a basic tool in itself, but also is the stepping stone to other important techniques in the lab going hand in hand Western blotting (WB). You can't have a good western blot without first having a good SDS gel. In this particular tips segment, we are discussing protein gel electrophoresis using precast gels and the parameters involved. Below are some pointers for optimizing your SDS-PAGE results.


1 Safety First

Please remember that typical voltages and currents used in electrophoresis are dangerous and possibly lethal. Make sure that all cables and connections are routinely inspected for cracks and other defects from normal wear and tear that result in exposed wires and replace immediately. Avoid inserting or removing high-voltage leads with both hands to prevent shunting electricity through the body and make sure to always handle those when the power supply is turned off. When disconnecting the system, first wait until the voltage and current meters reach zero, turn off the power supply, and then disconnect the gel box.


2 Choosing the Right Gel

Choosing the correct percentage gel is key. Rule of thumb, the larger the protein the lower percentage gel and vice versa. With that in mind, what is the size of your target? If you know your target's molecular weight then you can get away with one percentage, but if you don't, gradient gels are better for this purpose. Commonly used are 4-20% gradient gels that can cover a vast range of molecular weight sizes. Proteins ≥ 200 kDa will resolve better in 4-8% gels.


3 How Much to Load

Consider the concentration of your purified protein, lysate, or culture sample. Ideally, it is best to load ≤2 µg per well of a purified protein or ≤20 µg of a complex mixture like whole cell lysates if you are doing Coomassie stain only. Protein loading can be adjusted accordingly for more sensitive stains like silver and fluorescent staining or when doing WB where you can do lower amounts. Loading too little will result in an incomplete picture of your target such as any contaminants or accurate detection of your target protein. Overloading can result in smears or streaks due to protein precipitating or aggregating. In general, improper loading will lead to inconclusive results.


4 Timing

After samples are loaded, you want to begin to run your gel immediately after. Not doing so will result in your samples diffusing out of the wells. If you need to leave for a short amount of time, you can run your gel at low voltage to keep your samples from diffusing until your return. Now as for actually running your gel, follow manufacturer's recommendations of gel apparatus and pre-cast gel instructions. Typical conditions include runs at 100-150 volts for 40-60 minutes or until the dye front has reached the bottom of the gel. Letting it run too long will result in losing your lower molecular weight bands. Running too short and you will have poor resolution, especially in the low molecular weight range.


5 Heating Samples During Denaturation

Although not all samples need heating, it is critical for certain preparations like those containing membrane proteins. Thoroughly heating your sample not only completes the denaturing process but also ensures the dissociation of hydrophobic interactions, like those involving lipids. Not heating long enough results in incomplete denaturing, heating too long can cause aggregation. In general, heating at 95°C for 5 minutes should suffice. Following the denaturation step, spin down of samples at max speed for 2-3 minutes is critical to separate any particulates or aggregates that would otherwise interfere with gel loading and electrophoretic separation.


6 To Reduce or Non-Reduce

When considering protein molecules, which are built up of complex quaternary, tertiary, secondary, and primary structures, we want to denature our samples to its relative simplest structure for electrophoresis. While SDS helps to break these structures it also confers a negative charge to the amino acids giving a constant charge to mass ratio to all proteins and allowing separation to occur based on size. Nevertheless, SDS works well for secondary/tertiary structures but, for more complex structures like quaternary, we need to apply DTT (dithiothreitol) or βME (2-mercaptoethanol). These reducing reagents break disulfide bonds which are covalent and do not normally break using SDS alone. Both reducing agents work well, advantage of DTT is it has less of an odor than βME but breaks down faster. You can make your sample buffer with βME and have it freeze, thaw over and over due to its stable properties. If you are looking to analyze molecular weight complexes, you would be performing a non-reducing electrophoresis and would not use reducing agents.


7 Proteins Properties

One must consider the protein's properties when preparing the sample for SDS-PAGE. Typically, you add your sample of known concentration to some amount of SDS Sample buffer which is typically made up of SDS, glycerol, Tris-HCI pH 6.8, EDTA with or without reducing agent. In general, a 2X buffer stock can be used unless you have a much-diluted sample, in this case, would be beneficial to use a more concentrated stock (i.e., 5X or 6X) that allows you to load more samples in the same final volume. Proteins can also be further concentrated using trichloroacetic acid (TCA), acetone, or methanol at the expense of some net loss. Depending on the gel's well size, you want to keep volume in mind, leading up to our next tip.


8 Gel Loading

When loading your gel, it may be easier to use your standard tips that you use on a daily basis. However, by using gel loading tips you have more control of your sample. The tip is designed to go into the gels with narrow wells. Gel tips allow dispensing your sample accurately and precisely without the risk of overflowing (and cross-contaminating) into the next well. Always run gel electrophoresis markers along with your samples since these are not only necessary to determine accurately protein sizes but also serve as a control for the separation.


9 Gel Temperature

Maintaining a constant temperature between 10°C-20°C during separation is paramount in obtaining even migration of proteins. Otherwise, outer lanes will move slower than center lanes resulting in an artifact known as “smiling”. To prevent this negative effect, you can ensure efficient heat transfer by completely filling the outer chamber of the electrophoresis apparatus with SDS running buffer and constantly stirring the buffer with a magnetic stirrer. Alternatively, heat can be reduced by lowering the running current during separation.