How do I know the minimum concentration needed for measuring the size of my protein?
We are asked this question all the time when we are demonstrating the Zetasizer. Happily, if the Zetasizer software is installed (and it can be downloaded for free, by the way) there is a minimum concentration calculator integrated – so it’s simple! The calculator can be accessed from Tools – Calculators – Concentration Utilities – Minimum Concentration Calculator
Let’s look at a few examples to get an idea of some typical values for your samples. The left hand column of the table below lists a typical range of protein molecular weights, extending from 3 kDa to 250 kDa, which covers the majority of typical protein samples except for small peptides and large proteins. An estimated size for each weight of protein, measured by DLS, is shown in the middle column (both radius [r.nm] and diameter [d.nm] in nano meters). The sensitivity, listed in the right hand column, is the minimum concentration of the protein sample in mg/mL that is needed in order to achieve a result within a reasonable measurement duration. These values are for a standard Zetasizer Nano ZS, and also apply to the Zetasizer APS, microV and ZSP models. It really is as easy as 1, 2, 3!
How pure does my sample have to be ?
In principle, there is no requirement for protein samples measured using the Zetasizer to be highly purified. The main concern is that enough light reaches the detector so that the signal can be reliably analyzed. If the sample is so aggregated that light scattered by the larger aggregates overshadows that scattered by the monomer, then this will most likely lead to disappointment. In such cases, some centrifugation or filtration may resolve the problem; alternatively, increasing the sample concentration may lead to improved result quality.
The sensitivity calculator in the Zetasizer software also includes a section titled “Flow-Mode”. The concentration given applies to a typical one-column SEC chromatography setup (for example a Superdex 200) for the injection of 100 μL at the indicated concentration. For example for an antibody, an injection of 50 μg (100 μL * 0.5 mg/mL = 0.05 mg = 50 μg), see screen shot below. This would be valid for the Zetasizer microV, or Nano-S or Nano-ZSP when connected in flow mode to a chromatography system. Flow mode operation is also sometimes called ASEC (absolute size exclusion chromatography) and also referenced as DLS (dynamic light scattering), QUELS (quasi-elastic light scattering) or PCS (photon correlation spectroscopy) in flow. Both the hydrodynamic size and the scattering intensity of the eluting molecules is recorded in the chromatogram, as peaks elute from the size exclusion chromatography system.
How about the S90 system?
The traditional 90 degree scattering optics in the Zetasizer S90 (and the ZS90) requires a higher concentration in order to receive significant scattering intensity to acquire repeatable dynamic light scattering data. The minimum concentration calculator can also be used to find this sensitivity, and below is an example of a typical relationship for a protein close to the size of an antibody. The required concentration in the S90 is quite a bit higher, and that is why most protein researchers prefer the backscattering optics.
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