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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.

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Figure 1: The estimation of protein pI values using colloidal AFM-based force spectroscopy.
Figure 2: Estimation of pI values of model proteins.
Figure 3: Estimation of the pI value of cyprid footprint proteins using colloidal AFM-based force spectroscopy.

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Acknowledgements

The authors are grateful to the Agency for Science, Technology and Research (A*STAR) for providing financial support under the Innovative Marine Antifouling Solutions (IMAS) program.

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Contributions

G.J.V. supervised the project. D.J. and G.J.V. conceived the approach, evaluated the results and corrected the manuscript. S.G. conceived the approach, designed the experiments, analysed the data and wrote the paper. X.Z. designed the experiments and fabricated the surfaces. S.S.C.L. cultured the barnacle cyprids. T.H. contributed to the probe modification and discussion. S.L.M.T. evaluated and commented on the results. S.G. and X.Z. contributed equally to this work.

Corresponding authors

Correspondence to Dominik Jańczewski or G. Julius Vancso.

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The authors declare no competing financial interests.

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Guo, S., Zhu, X., Jańczewski, D. et al. Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample. Nature Nanotech 11, 817–823 (2016). https://doi.org/10.1038/nnano.2016.118

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