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Protein DLS result not as expected? Try these troubleshooting tips

Protein DLS result not as expected - troubleshooting tips

If your DLS result for a protein sample is not what you expected, it could be down to one of these common reasons.

Use this list to troubleshoot your DLS protein results fast.

These are the most common problems encountered when sizing proteins by DLS, and while many do have quick fixes these can sometimes simply mask or reduce the effect but not solve the underlying problem.

Making yourself aware of the different protein sizing techniques available, and the strengths and weaknesses associated with each, is the best option. With this knowledge you can pick the best analysis for your sample type, and ultimately save a lot of time.

1. The size reported is larger than I expected?

Possible cause: dust or other particulate(s) in the sample

Solutions:

  • If your instrument has an analysis mode which removes outliers check that this is set.

  • Check if your instrument allows each scan from a reading to be viewed separately – if there are only trace amounts of contamination it’s likely they would be detected in some but not all scans. The software may allow you to disregard some scans from the result, which can overcome this issue.

Possible cause: aggregation in the sample

Solutions:

  • Try to remove the aggregates by syringe filtration or centrifuge, then test the sample again. Remember that the intensity of light scattering is proportional to particle diameter to the 6th power, so even small amounts of aggregate or contaminants can give a big skew in results.

  • Alternatively, if you only have a very small volume of sample available and these options are not practical, dilution can reduce the relative concentration and therefore effect of aggregates. Note that dilution can affect aggregate stability, and could influence results due to concentration dependent sizing, so consider these factors when using this option.

  • Get a second opinion – test by another technique, ideally one not based on light scattering so this is not a factor. For example an MDS (microfluidic diffusional sizing) or gel electrophoresis size result could be used.

2. The size reported is smaller than I expected?

Possible cause: concentration dependent sizing

Solutions:

  • If samples are at very high concentration, multi-scattering effects can mean the light interacts with multiple particles before reaching the detector – losing intensity as it does. This results in an artificially small size being reported. Try diluting the sample to see if the size remains the same.
  • If the protein is one you will need to test frequently, testing a complete dilution series can help to find the optimum concentration to size this specific sample. Again remember that dilution can reduce the stability of aggregates, so approach with caution.

3. Multiple peaks are all not at the size expected?

Possible cause: solvent viscosity and refractive index

Solutions:

  • Check that these parameters are correctly set within the software. There may be the option to choose from standard solvents which will give a closer value.

  • If you are working in a unique formulation that you use frequently, it may be worth measuring the exact viscosity and refractive index experimentally. Often the options within DLS software are for pure solvents, and don’t account for the salts, reducing agents and other excipients that proteins, peptides and antibodies usually require.

Possible cause: scattering from non-protein elements

Solutions:

  • Minimize the use of detergents, surfactants and other excipients in the buffer so far as is possible. Dilution may also help to reduce their influence.

  • Get a second opinion – again using an orthogonal technique not based on light scattering could reveal more, or may be more appropriate for a sample where there are a large or varied number of additives present.

4. My protein of interest does not appear to be present?

Possible cause: peaks not resolving separately - your protein is "hidden"

Solutions:

  • Use a separation method, for example Size Exclusion Chromatography (SEC) to separate different species out, and then test again. It varies by instrument but most DLS tests need species to have a 3x to 5x difference in diameter to resolve separately – that’s around an 80 fold difference in molecular weight for proteins, so oligomers or even small aggregates can see this issue.

  • Check your instrument settings – some of the advanced modern instruments have settings to improve resolving power, such as using multi angle DLS.

  • Does your sample contain excess detergent micelles? These have been found to swamp the signal and make it hard to identify the protein separately. In this instance a technique that labels proteins only and specifically may be better – for example MDS or gel electrophoresis with a suitable dye.

If you're still not confident with your protein DLS results, get in touch with our application scientists here to discuss your samples and other techniques you could try.

  • Publications and resources

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    • Infographic

      Which protein sizing technique is right for me?

      This infographic lays out the key considerations you should think of when choosing a protein sizing technique.

    • Blog

      Sources of inaccuracy in protein aggregation measurement

      This blog explores the points to look out for when measuring protein aggregation through a variety of techniques that can reduce the accuracy of your results.

    • Application note

      Protein Size as an Indication of Structure

      Molecular weight (Mw) is a commonly used, and for many scientists a readily understood, parameter to describe the size of a protein or complex. Here we show how hydrodynamic radius (Rh) can be used in combination with Mw to provide insights into the shape and structure of proteins and illustrate how Mw alone may not always provide a complete picture.

    • Application note

      A comparison of Microfluidic Diffusional Sizing with Dynamic Light Scattering and Taylor Dispersion Analysis

      The established technologies of Dynamic Light Scattering (DLS) and Taylor Dispersion Analysis (TDA) are compared to Microfluidic Diffusional Sizing (MDS) for sizing proteins of varying molecular weights and at varying concentrations. We show that MDS offers comparable sizing of proteins over a range of sizes, and can provide consistent sizing to lower concentrations than the other techniques.

    • Blog

      Protein size - how do I measure it, and why is it important?

      An overview of why protein size matters, and what structural and functional information protein size can reveal. To understand proteins and their function, we have to understand the way they fold, aggregate and interact. Conformation is key to protein function and can be revealed by measuring size.

      Different methods for measuring protein size are summarised, and comparison is made, considering the method, range, cost and limitations of each technology.

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