57
When following any experimental protocol, it is
important to always do each of the steps the same way.
Preparing DLS samples is no exception.
Unless the purpose of your experiment is to measure
how your sample recovers from storage or a er
purification, you will need to remove contaminants and
pre-formed aggregates.
l For larger volumes of protein sample, start by
filtering with 0.02 µm cutoff filters.
l For smaller sample volumes or a er filtering,
centrifuge at ≥ 14.5 g for 10 minutes at 4 ᵒC before
loading the sample.
Once any aggregates or contaminants are removed,
you will be ready to run a quality assessment.
Consistent sample prep is key
There is no inherently "correct" r
H
. Every protein's
size is affected by the factors mentioned on Page 37.
Though there is usually an expected size for biological
samples, especially mAbs, there is always some
variation based on their environment, sequence,
and treatment. Creating good reference samples and
monitoring the variation in r
H
of derivative candidates
is a useful way to learn how environmental changes
affect the folding of a protein, but it is important to
also consider how the PDI and peak widths change
under different conditions.
Absolute vs relative size
