Top tips for optimum SDS-PAGE results

Top tips for optimum SDS-PAGE results

The process of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique in biochemistry, molecular biology and biotechnology. The method is commonly combined with antibody-based detection in Western blotting procedures and can be used in antibody application to differentiate proteins from more complex mixtures.

Below, we have assembled our top tips for getting the best results possible out of your SDS-PAGE procedures.

Should the sample wells be rinsed before loading the gel?

Yes, clearer results can be generated if the sample wells are rinsed and filled with water before the gel is loaded and before the buffer is placed into the tank. This technique can often lead to more precise results due to the discrepancy in densities between the water and tested sample, which is greater than the density discrepancy between the sample and the chosen buffer. We recommend using deionised water which can help to maintain stacking by reducing the chance of trailing ions from the buffer diffusing below the sample.

Can the use of reducing agents lead to better-prepared samples?

Yes, incorporating reducing agents, such as dithiothreitol, into sample preparation can lead to significantly clearer results. Reducing agents can be used to break existing intramolecular and intermolecular disulphide bonds which would otherwise impede the denaturisation process. The reducing agent breaks the bond by diminishing the S-S link of the two SH’s. This can lead to clearer bands as it lets the protein fully unfold and, in turn, re-establishes the relationship between molecular weight and migration.

What is the right quantity of protein to load?

The right quantity can depend on a number of factors, such as the culture sample, the purified protein and lysate, but there are aspects which can be considered across all SDS-PAGE experimentations. It is usually recommended that no more than 0.5 µg per band is loaded from cell lysates. Additionally, it may be beneficial to adjust the volume loaded when performing tests which require a higher level of sensitivity, for example when carrying out fluorescent staining procedures. Where overloading the wells can lead to streaks on the final product, underfilling can similarly lead to inconclusive results.

Can heating the samples lead to clearer results?

Yes, we recommend heating the samples once they have been mixed with a suitable buffer as it can boost the denaturing process. An adequate denaturing process is vital to reliably segregate proteins according to their molecular weight. Where it is not essential for all samples, it is particularly necessary for methods incorporating membrane proteins. Heating also guarantees the dissociation of hydrophobic interactions. Equally, heating for too long can cause protein aggregation. We recommend heating to 95℃, the optimal duration can vary between protocols but is usually between 2 – 10 minutes.

Can gel temperature effect migration and if so, should it be controlled?

Stringently keeping the gel temperature cool and constant can lead to clearer SDS-PAGE protein bands. Higher temperatures can affect the band shape and the quality of sample separation. Maintaining temperature is understandably a particular benefit when using temperature sensitive proteins which would otherwise degrade too quickly to visualise after either staining or Western Blot analysis. One tip is to place ice packs around the gel plates. An added benefit of this technique is that it can raise the buffer level in cases of shortage and sustain the inner tank volume. An ideal temperature range to aim for is between 10 – 20℃.

Are there any gel-related techniques which can optimise protein renaturation?

When performing protein renaturation or completing sequencing applications, the process can be aided by leaving the gel for a minimum of five hours post-polymerisation. Doing so will let the sample and components interact with the gel and limit the likelihood of a reaction occurring with the amino-terminal end of the peptide.

Is there an optimal time after loading when the SDS-PAGE process should commence?

Once the sample has been loaded, the process should be started immediately. This means that all methodological preparations, such as determining a voltage and placing the buffer in the tank, should be completed before loading the sample into the apparatus. If the process is not immediately started then the sample solution could diffuse out of the wells. Where it is essential to run the gel immediately, it is equally important to run it for the correct duration. Running the gel for too short a time could lead to poor resolution and unclear bands. Conversely, running the gel for too long could result in lower molecular weight bands being lost. In each case, specific centre and manufacturer guidelines should be followed; typically, the gel should be run immediately for approximately 50 minutes at 100 – 150 volts.

Whether SDS-PAGE is being used as a single procedure, in conjunction with ELISA, as part of a Western blot analysis or combined with another antibody-based detection method, these tips should improve the clarity of experimental results. In doing so, a research study could conserve valuable time, money and other vital resources.