Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample
Shifeng Guo , Xiaoying Zhu , Dominik Jańczewski , Serina Siew Chen Lee , Tao He , Serena Lay Ming Teo , G. Julius Vancso
AbstractProtein 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.
|Journal series||Nature Nanotechnology, ISSN 1748-3387|
|Publication size in sheets||0.5|
|Keywords in Polish||AFM, punkt izoelektryczny protein, LbL|
|Keywords in English||nanobiotechnology; sensors|
|ASJC Classification||; ; ; ; ;|
|Score|| = 50.0, 31-01-2020, ArticleFromJournal|
= 50.0, 31-01-2020, ArticleFromJournal
|Publication indicators||= 17; = 11; : 2016 = 7.649; : 2016 = 38.986 (2) - 2016=44.323 (5)|
* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.