Expansion of Light Scattering Service:

Dynamic Light Scattering Instrumentation

HPLC Size Exclusion Chromatography/Laser Light Scattering Determination of Native Protein Molecular Weights  (abbreviated as SEC-LS)that uses light scattering to determine the native molecular weights and sizes of proteins and their protein:protein complexes in solution.  The molecular weights are determined either during continuous-flow detection (in combination with size exclusion or other chromatography) or via a batch mode experiment.  Protein molecular weights may be determined quickly and accurately with an average error below 7% (over a range tested, i.e. from 7 to 480 kDa).

Light scattering (LS) is a non-invasive technique that provides the absolute mass (MW) and size (radius of gyration) of native macromolecules in solution (more information about light scattering  in Theory and about the service inHPLC Size Exclusion Chromatography/Laser Light Scattering Determination of Native Protein Molecular Weights).  Macromolecules can be analyzed at various pH values, ionic strengths and concentrations.  Light scattering studies can be carried out in either batch or chromatography mode.

Although molecular weights can be determined also via mass spectrometry and analytical centrifugation, only light scattering and analytical centrifugation monitor the properties of macromolecules in solution and provide information about the oligomeric state of the protein.  While a sedimentation equilibrium run may require 72 hours, a size exclusion chromatography/LS study can be completed in about an hour.  The comparatively short time of an LS run greatly facilitates carrying out the multiple studies that may be needed to determine the impact of protein concentration, ligands, pH.

More information about the service in:

Light Scattering Service - description of  the instrumentation used and sample requirements
Results for standard proteins  - compilation of data and results for standard proteins
Light Scattering Data Set   -   see an example of the extensive and completely interpreted data package you will receive back from us.

Introduction to Light Scattering  - general description of light scattering experiments

Light Scattering Theory  -  theory of light scattering and it's application to molecular weight determination

Invited Talk at 2002 ABRF Meeting "Determination of Molecular Masses of Proteins in Solution: Implementation of an HPLC Size Exclusion Chromatography and Laser Light Scattering Service in a Core Laboratory"  (*.pdf file)

Invited Talk at 2002 International Light Scattering Colloquium  "Analysis of protein complexes by size exclusion chromatography coupled with light scattering"  (*.pdf file)

Invited Talk at Wesleyan University, Middletown, CT, Feb. 2005 "Size Exclusion Chromatography Coupled with Light Scattering: Application to Study Proteins and Protein Complexes"

Invited Talk at Follow on Biologics Workshop, Dec. 14, 2005  "Light Scattering as a Tool for Assessing Protein Aggregates"  (*.pdf file)

Invited Talk at Impurities In Biomolecules organized by Institute for International Research, Nov. 8, 2006  "Monitoring & Predicting Biomolecular Aggregation Using Light Scattering"  (*.pdf file)

Invited Talk at BioProcess International™ Analytical and Quality Summit Impurities, Jun. 4-6, 2007  "Application of Light Scattering Techniques for Analysis of Oligomerization and Particle Formation  " (*.pdf file)
 
 

Publications that have reported SEC/LS data collected and analyzed at biophysical resource
[SEC/LS] and Dynamic LS
 
 

1. E. Folta-Stogniew and K.R. Williams (1999) “Determination of molecular masses of protein: implementation of a size exclusion chromatography/laser light scattering service in a core laboratory.” J. Biomol. Techniques, 10, 51-63.

2. P. D. Zamore, D. P. Bartel, R. Lehmann and J. R. Williamson| (1999) “The PUMILIO-RNA Interaction: A Single RNA-Binding Domain Monomer Recognizes a Bipartite Target Sequence” Biochemistry 38, 596-604

3. K. J. Lampi,  J. T. Oxford, H. Bachinger, T. R. Shearer, L. L. David and D. M. Kapfer (2002)  “Deamidation of Human ?B1 Alters the Elongated Structure of the Dimer” Exp. Eye Res. 72, 279-288

4. S. A. Robson, K. A. Michie, J. P. Mackay, E. Harry and G. F. King (2002)  ”The Bacillus subtilis cell division proteins FtsL and DivIC are intrinsically unstable and do not interact with one another in the absence of other septasomal components” Molecular Microbiology  44(3), 663–674

5. M. L. Bovee, K. S. Champagne, B. Demeler and C. S. Francklyn (2002) “ The Quaternary Structure of the HisZ-HisG N-1-(5’-Phosphoribosyl)-ATP Transferase from Lactococcus lactis” Biochemistry, 41, 11838-11846

6. T. Makagiansar, P. D. Nguyen, A. Ikesue, K. Kuczera, W. Dentler, J. L. Urbauer, N. Galeva, M. Alterman and T. J. Siahaan (2002) “Disulfide Bond Formation Promotes the cis- and trans-Dimerization of the ?-cadherin-derived First Repeat” J. Biol. Chem. 277, 16002–16010

7. G. P. Bertenshaw,  M. T. Norcum and J. S. Bond (2003) “Structure of Homo- and Hetero-oligomeric Meprin Metalloproteases”  J. Biol. Chem. 278, 2522–2532

8. Fraser,G. M.; Gonzalez-Pedrajo,B.; Tame, J. R.H.; Macnab,R. M. (2003) “Interactions of FliJ with the Salmonella Type III Flagellar Export Apparatus” The Journal of Bacteriology,  185, 5546-5554,

9. C. Keeler, P. S. Dannies and M. E. Hodsdon (2003) “The Tertiary Structure and Backbone Dynamics of Human Prolactin” J. Mol. Biol. 328, 1105–1121

10. Mitic,L. L.; Unger,V. M.; Anderson,J. M. (2003) “Expression, solubilization, and biochemical characterization of the tight junction transmembrane protein claudin-4.” Protein Science, 12, 218-227

11. D. Yernool, O. Boudker, E. Folta-Stogniew and E. Gouaux (2003) " Trimeric Subunit Stoichiometry of Glutamate Transporters." Biochemistry, 42, 12981-12988.

12. T. H. Scheuermann, E. Lolis and M. E. Hodsdon (2003) “Tertiary Structure of Thiopurine Methyltransferase from Pseudomonas syringae, a Bacterial Orthologue of a Polymorphic, Drug-metabolizing Enzyme”  J. Mol. Biol. 333, 573–585

13. Bruderer,R. M.; Brasseur,C.; Meyer,H. H.(2004)  “The AAA ATPase p97/VCP Interacts with Its Alternative Co-factors, Ufd1-Npl4 and p47, through a Common Bipartite Binding Mechanism.” J. Biol. Chem., 279, 49609-49616

14. Minamino,T.; Saijo-Hamano,Y.; Furukawa,Y.; Gonzalez-Pedrajo,B.; Macnab,R. M.; Namba,K. (2004) “Domain Organization and Function of Salmonella FliK, a Flagellar Hook-length Control Protein.” J. Mol.  Biol., 341, 491-502

15. Y. Wang and Ha,Ya (2004) “The X-Ray Structure of an Antiparallel Dimer of the Human Amyloid Precursor Protein E2 Domain” Mol. Cell  15, 343-353

16. M. E. Hahn and Muir TW (2004) “Photocontrol of Smad2, a multiphosphorylated cell-signaling protein, through caging of activating phosphoserines”  Angew. Chem. Int. Ed. Engl. 43, 5800-5803.

17. Thorne,M. E.; McQuade,K. L. (2004) “Heat-induced oligomerization of gp96 occurs via a site distinct from substrate binding and is regulated by ATP” Biochemical and Biophysical Research Communications, 323, 1163-1171

18. Chang,J.; Chen,J. ; Zhou,D.(2005) “Delineation and characterization of the actin nucleation and effector translocation activities of Salmonella SipC.” Mol. Microbiol., 55, 1379-1389

19. Yu, I-Mei, Gustafson , C. L. T., Jianbo, D., Burgner, J.W.,  Li, Z., Zhang, J. and Chen, J.  (2005) “Recombinant Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Forms a Dimer through Its C-terminal Domain” , J. Biol. Chem., 280, 23280-23286

20. Folta-Stogniew, E.(2006) “Oligomeric States of Proteins Determined by Size-Exclusion Chromatography Coupled with Light Scattering, Absorbance and Refractive Index Detectors”, Humana Press,  Methods in Molecular Biology, Nedelkov and Randall editors, 328, 97-112

21. Bunce,J.; Achila,D.; Hetrick,E.; Lesley,L.; Huffman,D, L. (2006) “Copper Transfer Studies between the N-terminal Copper Binding Domains One and Four of Human Wilson Protein.”, Biochim. Biophys. Acta, 1760, 907-912

22. Robles Lopez, SM, Hortua Triana, MA and Barbara H. Zimmermann (2006) “Cloning and preliminary characterization of the dihydroorotase from Toxoplasma gondii” , Mol. and Biochem. Parasitology, 148, 93-98

23. Stavropoulos, P, Blobel, G., Hoelz, A.  (2006) “Crystal structure and mechanism of the human lysine-specific demethylase LSD1.”  Nature Structural and Molecular Biology (in press)

24. Nelson, D., Schuch, R., Chahales, P. Zhu, S., Fischetti, V.A. (2006) “PlyC: the First Multimeric Bacteriophage Lysin.” Proc. Natl. Acad. Sci. U.S.A. (in press)

25.  Albright, R.A., Vazquez Ibar, J.-L., Kim, C. U., Gruner, S.M., Morais Cabral, J.H.  (2006) "Multiple conformations of the octameric ring structure of a RCK domain from a KtrAB K+ transporter” Cell (in press)
 
 

Comprehensive assessment of SEC/LS capabilities at:   http://abrf.org/JBT/1999/June99/jun99folta.html

Introduction to Light  Scattering	Light Scattering  Theory	Light Scattering  Data Set	Results for Standard Proteins	Light Scattering  Service	Biophysical resource  Front Page

For sample submission form go to HPLC SEC/Laser Light ScatteringSample Submission Page.
For pricing go to Service Charges  for HPLC SEC/Laser Light Scattering.

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