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Inline purification methods
Joint application SAXS & FPLC & Light
Scattering (SEC-SAXS/SLS)
Melissa Graewert
October 19th
The odd one out
What is SEC-SAXS, why does one need this?
What information is gained with light scattering?
How is SEC-SAXS done?
Experiment and data analysis
Sasha: Chromixs
Experiment
I(s)
s
X-ray beam
Experiment
I(s)
s
X-ray beam
What is SEC-SAXS/SLS, why does one need
this?
• Polydisperse samples
• Aggregates
• intrinsic property of the sample eg. monomer-dimer-
oligomer equilibrium or incomplete formation of
complexes
UV-Vis
BMSStarts HPLC
SEC-SAXS
BMS
UV-Vis
SEC-SAXS
Monomer
(65 kDa)Dimer
(122 kDa)
J. Synchrotron Rad. (2004)
Information extracted from the elution profile of an initially
polydisperse solution of commercial BSA. Column: SHODEX 402.5-
4F. Flow rate: 150 µl min−1. Injected volume: 5 µl at 88.8 g l−1. (a)
Chromatogram of the elution profile. The complex profile indicates
the presence of several species. The main peak, at 17.04 min,
corresponds to BSA monomer.
The SAXS instrument
at the Barkla
Laboratory of
Biophysics. The set-up
includes a Dectris
PILATUS 300K-20Hz
detector, three pin-
hole optics and Rigaku
FR-E+ Superbright X-
ray generator.
SEC-SAXS mode
SEC-SAXS/MALLS mode
Light absorbance: ~ c, ε
Light absorbance: ~ c, ε
Refraction: ~ c, dn/dc
dual cell, deflection design
Light absorbance: ~ c, ε
Scattering: ~ c, dn/dc, MW
Refraction: ~ c, dn/dc
SEC-SAXS/MALLS mode
monomer
mixture
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
Phospholipase B of Legionella pneumophila (Lpn PlaB)
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
— PlaB (batch, 4.5mg/ml)
— PlaB, tetrameric peak
lgI(
q),
a.u
.
q, nm-1
MWRALS = 230±15 kD
MWI(0 )= 225±15 kD
MWVol = 170±30 kD
MWDAMMIF= 203±30 kD
MWSEC ~ 100 kD
— PlaB (batch, 4.5mg/ml)
— PlaB, tetrameric peaklg
I(q
), a
.u.
q, nm-1
MWRALS = 230±15 kD
MWI(0 )= 225±15 kD
MWVol = 170±30 kD
MWDAMMIF= 203±30 kD
MWSEC ~ 100 kD
— PlaB (batch, 4.5mg/ml)
— PlaB, tetrameric peaklg
I(q
), a
.u.
q, nm-1
MWRALS = 230±15 kD
MWI(0 )= 225±15 kD
MWVol = 170±30 kD
MWDAMMIF= 203±30 kD
MWSEC ~ 100 kD
— PlaB (batch, 4.5mg/ml)
— PlaB, tetrameric peak
--- PlaB, dimeric peak
lgI(
q),
a.u
.
q, nm-1
Simultaneous Data Collection
Electron micrograph of Legionella
pneumophila wwww.wikimedia.org
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
host
pathogenLipolytic active
monomeric
PlaB
Activation
Via dimeric
state
Self protection
through inactive
tetrameric PlaB
Batch mode or SEC-SAXS mode
What is SEC-SAXS/SLS, why does one need this?
How is SEC-SAXS done
• Alternative strategy to study (moderatly) polydisperse samples
• Required sample amounts: at least 50 ul of > 5mg/ml
• Sufficient buffer
• Optimize your SEC run
• If possible collect batch sample as well
• Check for radiation damage, add 3% glycerol (if feasible)
