One of the advanced features in the Zetasizer Nano software is protein charge determination. In order to take advantage of the calculator, both size Dynamic Light Scattering (DLS) and electrophoretic mobility (zeta, ELS) data combine. In this post, we show how to use this facility to obtain the estimated charge of a protein.

#### Where is the protein charge calculator?

First, launch the classic Zetasizer Nano software. If you don’t have it installed, you can download it for free. Then go to Tools – Calculators – Protein Charge and f(κa). If that tab does not appear, you can enable the trial version of the advanced research features within the Zetasizer software. For a trial access go to Tools –> Options –> Feature Keys. See “How to try out the advanced protein features” for more detail.

The Protein Charge Calculator calculates the charge of a protein using the Ohshima equation for monovalent salts. It takes f(κa) from the Henry equation into account, for known size and ionic strength. We can measure the size by DLS and know the ionic strength from the preparation of the sample. (Alternatively, we could estimate the ionic strength from the conductivity):

#### What are the calculator setting parameters?

The Calculator Settings require several parameters to produce the protein charge. The first is the electrophoretic mobility as measured. For example with the diffusion barrier method and in the protein mobility mode with the classical Zetasizer Nano software.

The hydrodynamic radius is the size from dynamic light scattering. The calculator then asks for the ionic strength of the (mono-valent) buffer solution. The solution viscosity and temperature will often remain the defaults (for water, at room temperature).

#### Why the f(κa) results?

The right side of the Calculator contains the results from the entered input parameters. The temperature (in Kelvin) and dielectric constant (always for water) are first. 1/κ is the Debye screening length, κa the ratio of particle size to screening length. And, finally, f(κa) is calculated from the Henry equation using these parameters.

#### What are the protein charge results?

The reported zeta potential is the effective zeta potential using the correct Henry factor f(κa). As a result, the Calculated Z is the predicted average number of charges per protein molecule. If the number is negative, then the protein is negatively charged. If the number is positive, it is positively charged. In the example screen above, it would be minus 14.

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