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Laser
Light Scattering Determination of Native Protein Molecular Weights1 |
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
Results for standard proteins
compilation of data and results for standard proteins/font>
Light Scattering Data Set
see an example of the extensive and completely interpreted data package you will receive back from us.
More information about light scattering in:
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 talks:
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 beta 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.” Mol. Microbiol., 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 E-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.” J. Bacteriol., 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 Sci., 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. Wang, Y. and Ha, Y. (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 T.W. (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.” Biochem. Biophys. Res. Commun., 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., Diao, J., 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, S.M., Hortua Triana, M.A. and Barbara H. Zimmermann (2006) “Cloning and preliminary characterization of the dihydroorotase from Toxoplasma gondii.” Mol. Biochem. Parasitol., 148:93-98
23. Stavropoulos, P., Blobel, G., Hoelz, A. (2006) “Crystal structure and mechanism of the human lysine-specific demethylase-1.” Nat. Struct. Mol. Biol., 13:626-632
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., 103:10765-10770
25. Albright, R.A., Ibar, J.-L., Kim, C. U., Gruner, S.M., Morais Cabral, J.H. (2006) “The RCK domain of the KtrAB K+ transporter: multiple conformations of an octameric ring.” Cell, 126:1147-1159
Comprehensive assessment of SEC/LS capabilities at: http://abrf.org/JBT/1999/June99/jun99folta.html
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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.
Copyright © Ewa Folta-Stogniew. All rights reserved