Electrophoretic mobility measurements from proteins: How to protect proteins in an electric field

 

Protein-mobility-mode-measurement-proteins-between-electrodesWhat is the protein mobility mode?

When applying an electric field to a sample containing protein molecules, there are a few considerations to take into account to avoid “over-stressing” delicate biomolecules. The force a charged object experiences in an electric field is proportional to its charge times the electric field. Therefore the electric field strength (defined by the applied voltage divided by the distance between the electrodes, in V/cm) is more relevant than the absolute value of the applied voltage. In order to minimize detrimental field effects on delicate proteins, the field strength should be kept as low as possible, while still providing adequate energy to detect the electrophoretic movement on top of the random diffusion.

Potentially irreversible side effects like protein unfolding or denaturation may be caused by:

  • High field strength
  • Redox reactions at the electrode surface
  • Local Joule heating effects
  • Interference of additives (guanidine hydrochloride, surfactants, salts)

In order to minimize these adverse effects, the protein mobility measurement mode was developed for the Zetasizer Nano. In this special mode the electrophoretic mobility is determined differently from a standard zeta potential measurement. The protein-mobility measurement mode is not a direct analogue of the normal Zeta measurement mode with altered field settings, but it contains additional safeguards to avoid recording results from potentially aggregated (and thus damaged) proteins. There are four major advances incorporated into this unique mode:

  1. Gentle field settings: on-time, off-time and field strength are set from an algorithm developed from a detailed set of experiments that apply the appropriate settings to avoid Joule heating as much as possible. The dispersed phase is protected from thermal effects as much as possible.
  2. Selective recording of only the unaffected results: the algorithm de-selects aggregated sub-runs and introduces a delay between sub-runs to avoid Joule heating the sample and therefore possibly degrading the sample even faster. With this scheme we measure only the electrophoretic mobility of the native protein.
  3. Adjusted modulator frequency: Most proteins are rather small and therefore their frequency plot in a standard zeta measurement (for example for lysozyme) appears almost flat when using a standard 320Hz modulator frequency. The adjusted 1khz frequency in the protein mobility measurement is used to avoid this loss of resolution and provide an appropriate level of detection for the very fast diffusion in each sample time.
  4. Combination with diffusion barrier method: It is possible to use the diffusion barrier method and the protein mobility measurement together for additional safeguards of ultra-delicate molecules. If a sample degrades quickly, a shorter number of sub-runs may be specified, to save recording lots of aggregated, and therefore rejected, data, preventing the sample from unnecessary field exposure and stress.

In summary, the protein mobility measurement mode should be chosen to determine the electrophoretic mobility for the most accurate results and when working with delicate (or unknown stability) proteins.

What is electrophoretic mobility?

When an electric field is applied to a sample, any charged objects in the sample will be influenced by that field. The extra movement that particles exhibit as a result of them experiencing the electric field is called the electrophoretic mobility. Its typical units are μm·cm / V·s (micrometer centimeter per Volt second) since it is a velocity [μm/s] per field strength [V/cm]. The electrophoretic mobility is the direct measurement from which the zeta potential can be derived (using either the Smoluchowski/Debye-Hückel approximations or the complete Henry function F(κa) to get from the mobility to a zeta potential). For protein mobility applications, the advanced protein utilities can provide charge and f(κa).

How to access this software feature?

The protein mobility mode is only accessible when the advanced protein features have been enabled. These can be tested (30-day free trial) by going to

Tools -> Options -> Feature Keys -> Advanced Protein Features

and install the pre-populated License key that was generated for your software installation. The protein features will then be available for 30 days from activation, and there is no charge for this free trial.  Other benefits of the advanced protein features are discussed in ‘Key protein features in the Zetasizer software‘.

The protein mobility mode appears as a new measurement type, instead of only size and zeta measurements there will also be an additional mode as shown in the screen shot below. More specific details for the protein mobility mode are in the application note ‘Measurements of protein electrophoretic mobility using the Zetasizer Nano ZSP‘.

Measurement-type-protein-mobility-zetasizer

Previously

If you have any questions, please email me at ulf.nobbmann@malvern.com. Thanks! While opinions expressed are generally those of the author, some parts may have been modified by our editorial team.