A solid-phase assay for studying direct binding of progranulin to TNFR and progranulin antagonism of TNF/TNFR interactions.
The discovery that TNF receptors (TNFR) serve as the binding receptors for progranulin (PGRN) reveals the significant role of PGRN in inflammatory and autoimmune diseases, including inflammatory arthritis. Herein we describe a simple, antibody-free analytical assay, i.e., a biotin-based solid-phase binding assay, to examine the direct interaction of PGRN/TNFR and the PGRN inhibition of TNF/TNFR interactions. Briefly, a 96-well high-binding microplate is first coated with the first protein (protein A), and after blocking, the coated microplate is incubated with the biotin-labeled second protein (protein B) in the absence or presence of the third protein (protein C). Finally the streptavidin conjugated with a detecting enzyme is added, followed by a signal measurement. Also discussed in this chapter are the advantages of the strategy, key elements to obtain reliable results, and discrepancies among various PGRN proteins in view of the binding activity with TNFR.
Department of Orthopaedic Surgery, New York University Medical Center, Rm 1608, HJD, 301 East 17th Street, New York, NY, 10003, USA.
Qingyun
Q
Tian
Shuai
S
Zhao
Chuanju
C
Liu
D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D000077153:Progranulins; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects*; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D055767:Immobilized Proteins; D036341:Intercellular Signaling Peptides and Proteins; D000077153:Progranulins; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D001710:Biotin; D019809:Streptavidin