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Diffusion Barrier method – the practical details

24 November 2015 No Comment

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How to perform zeta potential measurement with the diffusion barrier method?

Diffusion barrier fig2The diffusion barrier method is particularly suitable for low sample volumes and high ionic strength buffers. A “plug” of the sample is carefully injected into the folded capillary cells which is filled with just the buffer (The buffer has to be the same buffer that the protein is prepared in, same conductivity, same pH, same additives, in order to match the sample and the diffusion barrier as closely as possible). In this setup, primarily the zeta potential is of interest, however the size can be determined as well.

Practical details are discussed in “The Diffusion Barrier Technique, Practical Aspects and Data interpretation” , the web presentation recording “Protein zeta potential measurements using Malvern’s new diffusion barrier method” as well as the application note “The Diffusion Barrier Technique for Accurate and Reproducible Protein Mobility Measurement“. A brief summary of the practical overview of the key components of this method is given below.

 

 

How is the sample introduced ?

Diffusion barrier fig3

The sample is gently injected into the buffer-filled DTS1070 capillary cell with a gel-loading tip. We have tested the Costar pipette tips (1-200µl, round, 0.5mm thick, Sigma cat:4853) and similar ones should work well.  For their use, gently push the gel-loading tip through one of the loading ports on the capillary cell until feel a “natural” end then inject 70µl at first (you will never go wrong with slug location in that case). As you get more familiar with the method, it is is easy to reduce the injection volume to 50µl, then eventually even down to 20µl. This can be practiced with either blue dextran solution, or by using the count rate monitor in the software (Tools – Count Rate Meter) to confirm that the scattering volume is within the sample slug.

There are  special instrument settings required to be able to measure zeta potential of protein samples in buffers with relatively high ionic strengths, The automatic mode in the zeta analysis software will reduce the applied voltage and for the most gentle handling of delicate sample, the analysis settings can be manually adjusted according to the table below, taken from the application note.

To access the settings during the Manual or SOP setup go to:

  • Measurement -> Measurement Duration -> Automatic -> Maximum number of runs
    Leave the minimum at 10 and select maximum according to table
  • Measurement -> Measurements -> Delay between measurements (sec)
    When repeating measurements, it is a good idea to let the sample rest for 30 seconds
  • Measurement -> Advanced -> Voltage -> Automatic Voltage Selection -> No
    Enter the suggested voltage according to the table

Diffusion barrier table

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.