I have been trying to measure the size of the human lysozyme protein with DLS. The instrument is Otsuka ELS-Z 1000. The upper and lower measurement size limits are 10 nm and 4000 nm respectively, and they cannot be changed in my SOP. However, after the measurement, a 'Recalculate' option is available which allows me to set a size lower than 10 nm. I have tried to measure the size for months now- I have changed buffers and concentrations, but to no use. The concentration is always optimal before and during measurement (~20,000 cps) but there are always insufficient data points, and the recalculate option cannot be used if there isn't sufficient data.
Measurement Conditions Temperature :25.0 (°C) Diluent Name :WATER Refractive Index :1.3328 Viscosity :0.8878(cP) Scattering Intensity :17957 (cps) Attenuator 1 :100.0 %
Measurement delay: 300 s
Scattering angle: 165 degrees
I clean the cell well (with soap solution, acetic acid 5%, DI water, ethanol), dry with Nitrogen air flow, use protein concentrations between 1-10 mg/ml, use KCl buffer at pH 2.5 (Lysozyme is most stable at pH less than 4/5 (I also tried PB, 1x PBS and 0.1x PBS) and use 0.2 micromolar membranes to avoid dust.
The measurements are successful with larger proteins, or when there is aggregation, but not with my samples. Any advice?
Ipsita Choudhury Filter all liquids with a 0.02 micron filter. A good rule in DLS is to filter down to the the level you want to measure.
Ipsita Choudhury From the calculator in the Zetasizer software you'd expect a size of just under 2 nm for a 15 kDa globular protein. Use the maximum concentration that you can to maximize the scattering.
Hello Ipsita Choudhury
you mention the mention the scattering intensity of your sample as about 20 kcps. What is the scattering intensity of just your buffer?
How does the correlation function look? Are there enough data points on the correlation function to detect a decay?
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