Shared Resources
Shared Resources

Biomolecular Interaction Analysis
Director: Munir Alam, PhD

Shared Resource Summary:

The DHVI Biomolecular Interaction Analysis Shared Resource, led by Munir Alam, provides specialized applications and support in Surface Plasmon Resonance (SPR) based biomolecular interaction analyses to basic and clinical researchers within the Duke Community. The facility offers state-of-the-art SPR BIAcore instruments for monitoring real time interaction analyses and diverse sets of measurements that include binding affinity, kinetics, epitope mapping and resolution of binding mechanism and structure. ­
 
BIAcore (GE Heathcare, Inc.;  www.biacore.com) is an optical biosensor based on SPR measurements for real time monitoring of biomolecular interactions without the need for labels. Standard applications include binding specificity, affinity and kinetics measurements of protein-protein, and protein-DNA interactions, and active concentration determination. In addition, the Facility offers expertise and methodologies for protein-lipid interactions that include analyses of antibody binding to phospholipids, synthetic liposomes, or intact virions and analyses of ligand (peptide-MHC monomer and tetramer, antibody) binding to detergent-resistant microdomains (“rafts”) from immune cell membrane. The SPR BIA Facility has developed protocols for monitoring and assessing binding avidity of antibody responses to immunogens and infectious agents using sera from immunized animals or patient samples. Multi-spot analyses of binding responses to a host of different antigens can be simultaneously monitored using the high-throughput, and multi-channel capability of the BIAcore A100.
 
The SPR Facility is also integrated with the DHVI Mass Spectrometry-Proteomics Core and offers applications in Mass Spectrometry based proteomics applications that include BIA-MS, in which a bound molecule is eluted and directly spotted on MALDI target plates for a MALDI mass spectrometry based identification of ligands for orphan receptors.

Personnel/Contact Information:

Director: S. Munir Alam, PhD
Office Phone: (919) 668-6372
Lab Phone: (919) 668-6995
Fax: (919) 684-5380
Email: alam0004@mc.duke.edu
Manager: Moses Sekaran, PhD 
Office Phone: (919) 668-6995
Lab Phone: (919) 668-6995
Fax: (919) 684-5380
Email: moses.sekaran@duke.edu
 
Location: 4042-4044 MSRB II, DUMC Box 103020, Durham, NC 27710
 

Getting Started:

  1. Please see the Common Tools sidebar to register to use facilities, or request a job, and to retrieve data. 

  2. Please schedule a consultation meeting and fill out the request for job form.
  3. For a schedule of fees for services, please contact DHVI Shared Resources business office.

Facilities and Instruments:

Services Provided:

  • Experimental protocol development for binding affinity and kinetics measurements   
  • Consultation and evaluation of binding kinetics data and resolution of binding mechanism
  • Screening and characterization of anti-lipid antibodies  
  • Design of high-throughput and multi-channel antibody specificity and binding avidity measurements
  • Early kinetic screening of hybridomas in culture supernatant for mAb selection
  • Monitoring of antibody responses in animal sera, or patient samples
  • Protein Identification from 1D or 2D gels (more info)
  • Peptide or protein molecular weight determination (more info)
  • De novo sequencing (more info)
  • Protein identification and quantitation by LC-MALDI (more info)
  • MALDI Imaging (more info)
  • BIA-MS for identification of ligands (more info)

Protocols and Methods:

  • Preparation of protein samples for SPR measurements
  • Preparation of synthetic lipids and liposomes
  • Isolation of detergent resistant membranes (DRM) from cell lines and primary immune cells
  • Binding kinetics of superantigen with TCR and MHC class II. Methods in Molecular Biology, 2003, 214:65-85.

Affiliated Centers:

Publications:

2006-2009
 
Dennison SM, Stewart SM, Stempel KC, Liao HX, Haynes BF, Alam SM. Stable docking of neutralizing HIV-1 gp41 membrane proximal external region monoclonal antibodies 2F5 and 4E10 is dependent on the membrane immersion depth of their epitope regions. J Virol. 2009 Jul 29. [Epub Ahead Of Print]
 
Go EP, Chang Q, Liao HX, Sutherland LL, Alam SM, Haynes BF, Desaire H. Glycosylation Site-Specific Analysis of Clade C HIV-1 Envelope Proteins. J Proteome Res. 2009 Jul 31. [Epub Ahead Of Print]
 
Go EP, Irungu J, Zhang Y, Dalpathado DS, Liao HX, Sutherland LL, Alam SM, Haynes BF, Desaire H. Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility. J Proteome Res. 2008 Apr;7(4):1660-74. Epub 2008 Mar 11.
 
Alam, S.M. et al. Human Immunodeficiency Virus Type 1 gp41 antibodies that mask membrane proximal region epitopes: Antibody binding kinetics, Induction, and Potential for regulation in acute infection. J. Virol., 2008, 82(1):115-125.
 
Zhang et al., Cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens. J. Virol., 2008, 82(14): 6869-68679.
 
Haynes & Alam. HIV-1 hides an Achilles’ heel in virion lipids. Immunity, 2008 28(1): 10-12.
 
Alam S.M., Scearce RM, Parks et al. Human immunodeficiency virus type 1 gp41 antibodies that mask membrane proximal region epitopes: antibody binding kinetics, induction, and potential for regulation in acute infection. J Virol. 2008; 82(1):115-25.
 
Alam et al. The Role of Antibody Polyspecificity and Lipid Reactivity in Binding of Broadly Neutralizing anti-HIV-1 Envelope Human Monoclonal Antibodies 2F5 and 4E10 to gp41 Membrane Proximal Envelope epitopes. J. Immunol. 2007, 178:4424-4435.
 
Staats et al. In Vitro and in vivo characterization of Anthrax anti-protective antigen and anti-lethal factor monoclonal antibodies after passive transfer in a mouse lethal toxin challenge model to define correlates of immunity. Infect. Immun., 2007, 75:5443-5442.
 
Wang et al., Incorporation of high levels of chimeric human immunodeficiency virus envelope glycoporteins into virus-like particles. J. Virol., 2007, 81(20):10869-78.
 
Liao et al. A group M consensus envelope glycoprotein induces antibodies that neutralize susbsets of subtype B and C HIV-1 primary viruses. Virology, 2006; 353(2):268-282.

Yu et al. Detection of Ebola virus envelope using monoclonal and polyclonal antibodies in ELISA, surface plasmon resonance and a quartz crystal microbalance immunosensor.  J. Virol Methods. 2006; 137(2):219-228.
 
Dennison et al., Neuronal SNAREs do ot trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion. Biophys. J. 2006, 90(5):1661-1675.
 
Lam et al.  Using microcantilever defelction to detect HIV-1 envelope glycoprotein gp120. Nanomedicine: Nanotechnology, Biology and Medicine. 2006, 2(4):222-229.

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