I am trying to measure the Zeta-potential and protein mobility of LDH A (332aa, 33kDa). Since we have a very limited amount of the protein, we decided to use the diffusion barrier technique with 0.5mg/ml LDH A suspension. We encounter a big problem with protein aggregation, thus getting unreliable results. I would like to measure at pH 4-10, but even test measurements at pH 7 (pI for LDH A is 8.1) would fail.
The software would give values for zeta-potential and mobility, but except for the size distribution, no graphs would be generated and l would constantly get the message that data quality is poor due to aggregation happening not only before the measurement, but during the measurement as well (shown by a steadily increasing PDI). I would always use the Auto mode and Monomodal settings, but the Zetasizer would occasionally even abort the measurement.
I tried the same sample preparation and measuring procedure with BSA, but the aggregation problem persists. Even after testing in different buffers (Tris, HEPES, PBS) and different agents to prevent aggregation (Tween20, TritonX100, sucrose, glycerol, L-arginine, guanidine hydrochloride protocol), the majority of the sample would still stay aggregated.
The question is, is there a more reliable way to prevent protein aggregation before and during measurement? I don’t expect a perfectly monodisperse sample but something that would get me closer to a more acceptable PDI.
Or, if possible, is there a way to bypass the results of the aggregated protein (even though it is the majority of it) and only use the values for single protein peaks? What I mean by that is that is, out of two peaks that appear in the intensity graph (one for single protein, one for aggregated protein), can the second peak be deleted out of the equation and only to one remain for calculations, no matter the intensity?
Hi Anela,
Starting with the most alarming points you raise, what error message did you get when the Zetasizer aborted the measurement? The instrument will do some optimisation checks at the start of a measurement and there are certain conditions it checks for before starting the measurement, so more information on the error would be helpful to enable a solution to be suggested.
What version of the Zetasizer are you using? The Zetasizer Nano ZSP has a higher power laser than the other variants meaning that is has better capability to measure proteins and other low scattering samples.
Also, your concentration may be a little low. The specification point for zeta potential measurements based on lysozyme (14kDa) is 1mg/ml. You should expect to be able to measure lower than that for your sample given that it is larger, but when using the diffusion barrier method there may be some degree of dilution as the sample diffuses over time, making measurements more challenging with a low volume of low concentration sample.
Are you running zeta potential measurements or protein mobility?
Both zeta potential and mobility are reported for both measurement types but the processing is different between them.
In a protein mobility measurement, the data is analysed as the measurement progresses and data that is expected to be attributed to the detection of stray aggregates is omitted from the final result, so even if some aggregation does occur, the results can still be reliable.
Additionally, the protein mobility measurement performs a size measurement before and after the mobility measurements to check on the state of the sample. If this shows aggregation, it does not indicate that the measured mobility is invalid (as per the processing discussed above), but that the sample is not suitable for repeat measurement.
Mono-modal measurements will not produce a zeta potential distribution as this data is calculated from the Slow Field Reversal part of an applied waveform in a General purpose measurement. This is omitted from a mono-modal measurement for samples that are either fragile or suspended in a high concentration solution in order to reduce the possible effects of Joule heating.
Hello Arnela, a few additional comments, which may help.
Concerning the missing zeta distribution, the mean zeta is still available and this is expected for high conductivity measurements:
http://www.materials-talks.com/blog/2017/09/05/what-is-the-zeta-deviation/#nozeta
If you are concerned with sample amount and aggregation, it may be best to use DLS only and not measure zeta potential. The low volume disposable cuvettes ZEN0040 or the really low volume quartz cuvettes ZEN2112 are ideal for this. For DLS, you can potentially “eliminate” the large size peak by following the concept discussed in
http://www.materials-talks.com/blog/2016/06/15/solvent-peaks-how-to-get-rid-of-the-buffersaltadditive-dls-contribution/
It may be best if you send the data file (or a relevant selection of records) http://www.materials-talks.com/blog/2014/08/19/faq-how-can-i-submit-a-data-file-to-the-help-desk/ to Malvern for specific troubleshooting.
Thank you very much for your answers!
Yes I was using the Zetasizer Nano ZSP for the measurements and have changed the settings to general purpose to get a zeta-potential distribution. I am measuring protein mobility to follow the aggregation process. I also used the settings described in materials-talks (thanks Ulf!).
Still my question is, can I assume that the zeta-potential measurement I get for my mostly aggregated protein sample is the same I would get for a monodisperse, non-aggregated sample? What diffenrence can I expect for aggregated proteins, how to compare the protein mobility of the same protein but with different aggregation status? Any additional papers I could check?
Hi Anela,
From experience, the zeta-potential of aggregated vs non-aggregated proteins is usually different, which might not be too surprising if we imagine a protein molecule unfolding as an aggregation mechanism.
As well as doing size measurements of your sample to check for aggregates, you can get an indication on whether aggregates are detected in a zeta measurement by looking at the frequency plot. For a protein, which is small and diffusing relatively quickly, the frequency plot will be quite broad. An example of this is shown below, from
Corbett and Jack, Measuring protein mobility using modern microelectrophoresis, Colloids and Surfaces A, 376(2011)31-41 .
Can anyone suggest what solvent to use for Ti-Mg alloy to be used in zetasizer?? is this the accurate method of measuring particle size range for metal powders??
Adil Muneeb
I think it would be good practice to start your own discussion, particularly as your question is somewhat different.
In short, if you have a dry powder, in general this type of material will be better suited to a laser diffraction measurement.
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