SLIDE 1
Inline purification methods
Joint application SAXS & FPLC & Light Scattering (SEC-SAXS/SLS)
Melissa Graewert October 19th
The odd one out
Inline purification methods Joint application SAXS & FPLC & - - PowerPoint PPT Presentation
Inline purification methods Joint application SAXS & FPLC & - - PowerPoint PPT Presentation
Inline purification methods Joint application SAXS & FPLC & Light Scattering (SEC-SAXS/SLS) Melissa Graewert October 19 th The odd one out What is SEC-SAXS, why does one need this? What information is gained with light
SLIDE 2
SLIDE 3
- 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
SLIDE 4
- Experiment
I(s) s X-ray beam
SLIDE 5
- Experiment
I(s) s X-ray beam
SLIDE 6
- What is SEC-SAXS/SLS, why does one need
this?
- Polydisperse samples
- Aggregates
- intrinsic property of the sample eg. monomer-dimer-
- ligomer equilibrium or incomplete formation of
complexes
SLIDE 7
UV-Vis
BMS
Starts HPLC
SEC-SAXS
SLIDE 8
BMS
UV-Vis
SEC-SAXS
SLIDE 9
Monomer (65 kDa) Dimer (122 kDa)
SLIDE 10
- J. Synchrotron Rad. (2004)
SLIDE 11
SLIDE 12
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.
SLIDE 13
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.
SLIDE 14
- SEC-SAXS mode
SLIDE 15
- SEC-SAXS/MALLS mode
SLIDE 16
Light absorbance: ~ c, ε
SLIDE 17
Light absorbance: ~ c, ε Refraction: ~ c, dn/dc dual cell, deflection design
SLIDE 18
Light absorbance: ~ c, ε Scattering: ~ c, dn/dc, MW Refraction: ~ c, dn/dc
SLIDE 19
- SEC-SAXS/MALLS mode
SLIDE 20
monomer mixture
SLIDE 21
SLIDE 22
SLIDE 23
SLIDE 24
SLIDE 25
- M.O.S.E.S. (Microsplitting for Online Separation,
Extended characterization and SAXS analysis)
Phospholipase B of Legionella pneumophila (Lpn PlaB)
SLIDE 26
- M.O.S.E.S. (Microsplitting for Online Separation,
Extended characterization and SAXS analysis)
— PlaB (batch, 4.5mg/ml) — PlaB, tetrameric peak lg I(q), a.u. q, nm-1
SLIDE 27
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 lg I(q), a.u. q, nm-1
SLIDE 28
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 lg I(q), a.u. q, nm-1
SLIDE 29
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
lg I(q), a.u. q, nm-1
SLIDE 30
- Simultaneous Data Collection
SLIDE 31
Electron micrograph of Legionella pneumophila wwww.wikimedia.org
- M.O.S.E.S. (Microsplitting for Online Separation,
Extended characterization and SAXS analysis)
host pathogen Lipolytic active monomeric PlaB Activation Via dimeric state Self protection through inactive tetrameric PlaB
SLIDE 32
- Batch mode or SEC-SAXS mode
SLIDE 33
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)