Asymmetric flow FFF and Composition Gradient/Light Scattering System

Summary

Principal Investigator: EWA JOANNA FOLTA-STOGNIEW
Abstract: [unreadable] DESCRIPTION (provided by applicant): Funding is requested for state-of-the-art asymmetric flow Field-Flow Fractionation (AFFF) and automated composition gradient (CG) syringe delivery systems that would share light scattering (LS), refractive index, absorbance, and fluorescence detectors. AFFF would be used when more than one size macro- molecular complex is expected while CG would deliver to the same detectors samples that are solutions of purified proteins of various compositions to enable the evaluation of equilibria for homo- or hetero-associations. AFFF is a single phase chromatography technique. High-resolution separation by size is achieved within a very thin flow against which a perpendicular force field is applied. With AFFF separation there is no column medium to interact with the samples. The entire separation is gentle, rapid, and non-disruptive - without a stationary phase that may degrade, or other wise alter the sample. AFFF is capable of fractionating samples ranging in size from 1nm to >20 microns. The combination of AFFF as a fractionation step and light scattering (LS) as a detection method allows sample fractionation and determination of size and molar mass in a single experiment: the molar mass is determined from a static LS measurement and the hydrodynamic radius is determined from AFFF elution time and from dynamic LS measurements. The coupling of light scattering measurement to a fractionation step provides the most straightforward and cost effective method to determine the molar masses and oligomeric states of a wide range of diverse macromolecules including native or modified proteins and their complexes, nucleic acids, conjugated proteins, liposomes, and polysaccharides. In contrast to sizing by ultracentrifugation, fractionation and sizing on AFFF/LS allows the facile collection of fractions for further analyses (e.g., biological activities, electron microscopy, and mass spectrometry). The requested AFFF/CG/LS system is versatile and would support 23 projects directed by 13 Yale investigators and 10 investigators from 8 other institutions including Columbia, Harvard, Pennsylvania State, Purdue, and Rockefeller University. These 23 investigators propose to use AFFF/GC/LS to advance a diverse range of biomedical research on pancreatitis, Salmonellosis (which continues to be a major world-wide health concern and has an estimated annual economic impact on the U.S. of $3 billion), metalloproteases involved in acute renal injury or intestinal inflammation, dopamine transporters, prolactin receptor, intercellular junctions, remodeling complexes at locus control regions, nuclear pore complex, infection by group A streptococci, and the development of advanced drug delivery systems utilizing nano and microparticles. Progress in this research will likely advance our knowledge of how best to understand, prevent, and treat human diseases. If this grant application is funded, the AFFF/CG/LS system would be unique to Yale University and neighboring academic institutions, and it surely would make a unique contribution to biomedical research that would extend far beyond Yale University. [unreadable] [unreadable] [unreadable]
Funding Period: 2007-04-01 - 2008-03-31
more information: NIH RePORT

Top Publications

  1. pmc Keck Foundation Biotechnology Resource Laboratory, Yale University
    Kathryn L Stone
    Keck Foundation Biotechnology Resource Laboratory, Yale University, 300 George Street, New Haven, Connecticut, USA
    Yale J Biol Med 80:195-211. 2007
  2. pmc Asymmetric friction of nonmotor MAPs can lead to their directional motion in active microtubule networks
    Scott Forth
    Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
    Cell 157:420-32. 2014
  3. pmc The number and location of EF hand motifs dictates the calcium dependence of polycystin-2 function
    Ivana Y Kuo
    2B E E, Department of Pharmacology, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06520 8066, USA
    FASEB J 28:2332-46. 2014
  4. pmc Oligomerization and higher-order assembly contribute to sub-cellular localization of a bacterial scaffold
    Grant R Bowman
    Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, 94305, USA
    Mol Microbiol 90:776-95. 2013
  5. pmc Marking and measuring single microtubules by PRC1 and kinesin-4
    Radhika Subramanian
    Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
    Cell 154:377-90. 2013
  6. pmc MIF intersubunit disulfide mutant antagonist supports activation of CD74 by endogenous MIF trimer at physiologic concentrations
    Chengpeng Fan
    Department of Pharmacology, Yale University, New Haven, CT 06510, USA
    Proc Natl Acad Sci U S A 110:10994-9. 2013
  7. pmc Biochemical analysis of hypermutation by the deoxycytidine deaminase APOBEC3A
    Robin P Love
    Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
    J Biol Chem 287:30812-22. 2012
  8. pmc Concentration-dependent oligomerization and oligomeric arrangement of LptA
    Jacqueline A Merten
    Department of Biophysics, Medical College of Wisconsin, 8701 WatertownPlank Road, Milwaukee, WI 53226, USA
    Protein Sci 21:211-8. 2012
  9. pmc Recognition of the F&H motif by the Lowe syndrome protein OCRL
    Michelle Pirruccello
    Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA
    Nat Struct Mol Biol 18:789-95. 2011
  10. pmc Intensity of deoxycytidine deamination of HIV-1 proviral DNA by the retroviral restriction factor APOBEC3G is mediated by the noncatalytic domain
    Yuqing Feng
    Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
    J Biol Chem 286:11415-26. 2011

Scientific Experts

  • Kenneth R Williams
  • Pietro V De Camilli
  • Jacalyn M Green
  • Radhika Subramanian
  • Tarun M Kapoor
  • Ivana Y Kuo
  • Scott Forth
  • Grant R Bowman
  • Chengpeng Fan
  • Jacqueline A Merten
  • Robin P Love
  • Ewa Folta-Stogniew
  • Seth A Darst
  • Linda Chelico
  • Yuqing Feng
  • Michelle Pirruccello
  • Kathryn L Stone
  • Edward T Petri
  • Andjelka Celic
  • Kuo Chiang Hsia
  • Barbara E Ehrlich
  • Rachel Corbin
  • Camille Keeler
  • Yuta Shimamoto
  • Michael E Hodsdon
  • Elias J Lolis
  • Eseosa Ighodaro
  • Luis R Comolli
  • J Patrick Loria
  • Mansoor Ali Syed
  • Deepa Rajasekaran
  • Lin Leng
  • Vineet Bhandari
  • Lei Tan
  • Lucy Shapiro
  • Adam M Perez
  • Richard Bucala
  • Jerod L Ptacin
  • Shih Chieh Ti
  • Candice S Klug
  • Kathryn M Schultz
  • Huixin Xu
  • Laura E Swan
  • Elizabeth A Campbell
  • Ronald A Milligan
  • Matthew J Bick
  • Christopher P Arthur
  • Elizabeth M Wilson-Kubalek
  • Yong Kong
  • Gregory G Blasko
  • Erol Gulcicek
  • Zhao Hongyu
  • Mary B LoPresti
  • Janet K Crawford
  • Robert D Bjornson
  • P John Flory
  • Walter J McMurray
  • Nancy A Williams
  • Aiping Lin
  • Shrikant M Mane
  • James I Elliott
  • Joseph D Deluca
  • Terence L Wu
  • Margaret M Elliott
  • Ewa J Folta-Stogniew
  • J Myron Crawford
  • Renee Cofrancesco
  • Can Bruce
  • Irina R Tikhonova
  • TuKiet T Lam
  • Sheila Westman
  • Christopher M Colangelo
  • Nancy C daSilva
  • Ji Y Lee
  • Nicholas J Carriero

Detail Information

Publications12

  1. pmc Keck Foundation Biotechnology Resource Laboratory, Yale University
    Kathryn L Stone
    Keck Foundation Biotechnology Resource Laboratory, Yale University, 300 George Street, New Haven, Connecticut, USA
    Yale J Biol Med 80:195-211. 2007
  2. pmc Asymmetric friction of nonmotor MAPs can lead to their directional motion in active microtubule networks
    Scott Forth
    Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
    Cell 157:420-32. 2014
    ..Our findings reveal how nonmotor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures...
  3. pmc The number and location of EF hand motifs dictates the calcium dependence of polycystin-2 function
    Ivana Y Kuo
    2B E E, Department of Pharmacology, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06520 8066, USA
    FASEB J 28:2332-46. 2014
    ..These results suggest that the number and location of calcium-binding sites in the EF hand senses the concentration of calcium required for PC2 channel activity and cellular function...
  4. pmc Oligomerization and higher-order assembly contribute to sub-cellular localization of a bacterial scaffold
    Grant R Bowman
    Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, 94305, USA
    Mol Microbiol 90:776-95. 2013
    ..Thus, PopZ undergoes multiple orders of self-assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter. ..
  5. pmc Marking and measuring single microtubules by PRC1 and kinesin-4
    Radhika Subramanian
    Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
    Cell 154:377-90. 2013
    ..Our findings suggest how biochemically similar microtubules can be differentially marked, based on length, for selective regulation during the formation of specialized arrays, such as those required for cytokinesis...
  6. pmc MIF intersubunit disulfide mutant antagonist supports activation of CD74 by endogenous MIF trimer at physiologic concentrations
    Chengpeng Fan
    Department of Pharmacology, Yale University, New Haven, CT 06510, USA
    Proc Natl Acad Sci U S A 110:10994-9. 2013
    ..This conclusion has implications for therapeutic development. ..
  7. pmc Biochemical analysis of hypermutation by the deoxycytidine deaminase APOBEC3A
    Robin P Love
    Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
    J Biol Chem 287:30812-22. 2012
    ..That APOBEC3A was able to deaminate dsDNA undergoing transcription suggests a genomic cost of a deamination-based retroviral restriction system...
  8. pmc Concentration-dependent oligomerization and oligomeric arrangement of LptA
    Jacqueline A Merten
    Department of Biophysics, Medical College of Wisconsin, 8701 WatertownPlank Road, Milwaukee, WI 53226, USA
    Protein Sci 21:211-8. 2012
    ....
  9. pmc Recognition of the F&H motif by the Lowe syndrome protein OCRL
    Michelle Pirruccello
    Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA
    Nat Struct Mol Biol 18:789-95. 2011
    ..Our study predicts the existence of other OCRL binding partners and shows that the perturbation of OCRL interactions has a crucial role in disease...
  10. pmc Intensity of deoxycytidine deamination of HIV-1 proviral DNA by the retroviral restriction factor APOBEC3G is mediated by the noncatalytic domain
    Yuqing Feng
    Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
    J Biol Chem 286:11415-26. 2011
    ..The data demonstrate that the balance between the jumping and sliding of Apo3G is needed for efficient mutational inactivation of HIV-1...
  11. pmc Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein
    Radhika Subramanian
    Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA
    Cell 142:433-43. 2010
    ..Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish crosslinks that selectively "mark" antiparallel overlap in dynamic cytoskeletal networks...
  12. pmc Purification and characterization of the folate catabolic enzyme p-aminobenzoyl-glutamate hydrolase from Escherichia coli
    Jacalyn M Green
    Department of Biochemistry, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA
    J Bacteriol 192:2407-13. 2010
    ..08 microM and a specific activity of 63,300 +/- 600 nmol min(-1) mg(-1). Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway...