I'm looking at the aggregation of my protein sample using DLS. Unfortunately, my buffer (20mM HEPES) also results in a set of peaks. These are at approximately 1 and 1000 d.nm. The lowest peak disappears when protein is added to the sample, which I believe is due to 'intensity' giving more weight to larger particles. The peaks at sizes of 1000 to 10000 d.nm do not disappear and I'm not sure what to do with it. Is there a way to eliminate noise from my buffer?
Thanks in advance!
The buffer should have very weak intensity and should only show a peak at the smaller size. The larger size peak is probably an analysis artifact. You may be able to get rid of it by changing some parameter setting in the analysis. If that doesn't help, centrifuge the buffer (16,000 g for 10 min) to get rid of any dust that might be in it.
The protein signal, being much stronger than that of the buffer, will overwhelm the buffer peak and it will probably be undetectable. The very large size peak in the presence of the protein could be due to aggregates or particulates. That data is meaningful and should not be ignored, but it is worth remembering that larger particles scatter much, much more strongly than smaller ones, so the large size peak in intensity mode may reflect a very small amount of aggregate. If you centrifuge the sample to pellet the aggregate, the large size peak should be reduced.
If you can't resolve a peak corresponding to the expected size of the protein in mass mode (rather than intensity), then you would conclude that your protein is aggregated.
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