Protein Crystallography and
Biophysics Centre (BiophysX)
Institute of Structural and Molecular Biology
(ISMB)
Birkbeck College / University College London
SEC-MALS
(Agilent 1100, Wyatt
DAWN 8+, Wyatt Optilab
T-rex)
Size Exclusion
Chromatography coupled with
Multi-Anlge Static Light
Scattering (SEC-MALS) provides
direct information on the
molecular weight of the sample,
so it can identify aggregates or
oligomerization, characterize
large complexes and generally
assess the homogeneity of a
sample. Using in addition UV
detector, each species can be
dissected into different
components, particularly
glycosylation or bound
detergents. SEC-MALS is
particularly powerful for
optimizing the detergent content
on membrane proteins as also for
studying protein/DNA/RNA
complexes and glycosylated
proteins.
Set up and Sample Requirements
A Size Exclusion column usually of a
10/300 size is ideal for the current set
up. A 3.2/300 column can also provide
excellent results if the sample quantity
is low
Volume: typically 100 μl for a 10/300
column. The injector has a range 0.1 -
100 μl therefore the injection volume
can be adjusted depending on the actual
parameters for each experiment (Volumes
lower than 20 μl are not recommended
though).
Concentrations: For the 10/300 columns
1 mg/ml is a reasonable approximation.
10-fold lower concentrations have worked
for the 2.3/300 column.
Sample purity: purity is very
important, since aggregates or other
impurities can affect significantly the
signal.
Buffers need to be freshly made,
filtered and degassed.
A fraction collector is installed as
default in the system.
