title: Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample.
abstract: Protein charge at various pH and isoelectric point (pI) values is important in understanding protein function. However, often only trace amounts of unknown proteins are available and pI measurements cannot be obtained using conventional methods. Here, we show a method based on the atomic force microscope (AFM) to determine pI using minute quantities of proteins. The protein of interest is immobilized on AFM colloidal probes and the adhesion force of the protein is measured against a positively and a negatively charged substrate made by layer-by-layer deposition of polyelectrolytes. From the AFM force-distance curves, pI values with an estimated accuracy of ±0.25 were obtained for bovine serum albumin, myoglobin, fibrinogen and ribonuclease A over a range of 4.7-9.8. Using this method, we show that the pI of the 'footprint' of the temporary adhesive proteins secreted by the barnacle cyprid larvae of Amphibalanus amphitrite is in the range 9.6-9.7.
authors.affiliation: Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 2 Fusionopolis Way, Innovis, No. 08-03, Singapore 138634, Singapore.
authors.forename: Shifeng
authors.initials: S
authors.lastname: Guo
authors.affiliation: Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 2 Fusionopolis Way, Innovis, No. 08-03, Singapore 138634, Singapore.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 2 Fusionopolis Way, Innovis, No. 08-03, Singapore 138634, Singapore.|Laboratory of Technological Processes, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland.
authors.forename: Dominik
authors.identifier: 0000-0002-5466-6444
authors.initials: D
authors.lastname: Jańczewski
authors.affiliation: Tropical Marine Science Institute, National University of Singapore, 18 Kent Ridge Road, 119227 Singapore, Singapore.
authors.forename: Serina Siew Chen
authors.initials: SS
authors.lastname: Lee
authors.affiliation: Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 2 Fusionopolis Way, Innovis, No. 08-03, Singapore 138634, Singapore.
authors.forename: Tao
authors.initials: T
authors.lastname: He
authors.affiliation: Tropical Marine Science Institute, National University of Singapore, 18 Kent Ridge Road, 119227 Singapore, Singapore.
authors.forename: Serena Lay Ming
authors.initials: SL
authors.lastname: Teo
authors.affiliation: Institute of Chemical and Engineering Sciences A*STAR, 1 Pesek Road, Jurong Island, 627833 Singapore, Singapore.|MESA+ Institute for Nanotechnology, Materials Science and Technology of Polymers, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands.
authors.forename: G Julius
authors.initials: GJ
authors.lastname: Vancso
mesh_terms: D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000032:analysis* / Q000737:chemistry*; D007526:Isoelectric Point; D018625:Microscopy, Atomic Force / Q000379:methods; D012710:Serum Albumin, Bovine; D013499:Surface Properties; D001468:Thoracica / Q000737:chemistry
chemical_list: D055767:Immobilized Proteins; D012710:Serum Albumin, Bovine