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Protocol
Using Magnetic Tweezers to Unravel the Mechanism of the G-quadruplex Binding and Unwinding Activities of DHX36 Helicase
Single-molecule manipulation methods are useful techniques to probe the interactions of proteins and nucleic acid structures. Here, we describe the magnetic tweezers-based single-molecule investigation of the ...
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Protocol
Luminex-Coupled EliFACS: A Multiparametric Method to Enumerate and Functionally Characterize Antigen-Specific T cells in Human Peripheral Blood
We describe a Luminex-coupled EliFACS assay that integrates multiplexing technology, enzyme-linked immunospot (ELISPOT), and intracellular cytokine FACS staining for the detection of multiple parameters of ant...
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Protocol
High-Throughput Multimodal Strong Anion Exchange Purification and N-Glycan Characterization of Endogenous Glycoprotein Expressed in Glycoengineered Pichia pastoris
The secretory pathway of the yeast Pichia pastoris has been engineered to produce complex human-type N-glycans (Choi et al., Proc Natl Acad Sci USA 100:5022–5027, 2003; Hamilton et al., Science 301:1244–1246, 200...
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Protocol
Characterization of N-Linked Glycosylation on Recombinant Glycoproteins Produced in Pichia pastoris Using ESI-MS and MALDI-TOF
The production of recombinant therapeutic glycoproteins is an active area of research and drug development. Typically, improvements in therapeutic glycoprotein efficacy have focused on engineering additional N...
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Protocol
N-Linked Glycan Characterization of Heterologous Proteins
Our laboratory has focused on the re-engineered of the secretory pathway of Pichia pastoris to perform glycosylation reactions that mimic processing of N-glycans in humans and other higher mammals (1,2). A report...
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Protocol
Separation of Glycoproteins by Capillary Isoelectric Focusing
Capillary isoelectric focusing (cIEF) has now become a somewhat routine, accepted mode of operation in capillary electrophoresis (CE). We and others have published several reviews of cIEF (1–14). More and more ap...
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Protocol
Synthesis of Coumarin-Based, Esterase-Sensitive Cyclic Prodrugs of Opioid Peptides with Enhanced Membrane Permeability and Enzymatic Stability
With the discovery of an increasing number of biologically active peptides and peptide mimetics (1–3), there is a pressing need for the development of strategies to deliver these biologically active compounds to ...