Technical Support

I27O™ AFM Reference Protein Data Sheet

Catalog No. 0304

Product Description

I27O™ is a 94 kDa synthetic polyprotein composed of eight repeats of the I27 domain of human fibronectin. It is used for the calibration of and as a reference for force extension experiments using atomic force microscopes. It is supplied in a ready-to-use solution. Single molecule measurements give up to eight saw-tooth force-extension curves with the specifications below. The protein has durable elasticity that allows for repeated unfolding and refolding.

Product Specifications
Protein	100µg
Shipping	shipped on dry ice
Long-Term Storage	2 years at - 80°C Aliquot to avoid repeated freezing and thawing.
Short-Term Storage	1 month at 4°C
Formulations	In SSI Buffer: 50 mM Na2HPO4, 300 mM NaCl, 20 mM imidazole, pH 8.0

Technical Information (force extension measurements)
Distance between peaks	24.1 ± 0.34nm
Force Peaks	204 ± 26pN
Persistence Length	0.39 ± 0.07nm
Contour length	28.4 ± 0.34nm

Instructions for Use

1.	Dilute the stock protein solution to a concentration of 100µg/ml in PBS pH 7.3 buffer and apply the sample to freshly evaporated gold coverslips.
2.	Attach coverslips to the piezoelectric positioner stage.

The I27O™ was engineered with an amino-terminal hex-His peptide sequence and carboxy-terminal di-Cys. This confi guration is hypothesized to allow the protein molecules to attach to the coverslip at the carboxy-terminal leaving the amino-terminus free to attach to the silicon nitride cantilever tip, when the tip is brought in close proximity to the absorbed protein. It is this model which is believed to account for the high frequency (>50%) of observing all eight domain extensions after each retraction as well as the ability to perform extension and retraction experiments on a single molecule.

Material Safety Data

FOR RESEARCH USE ONLY. NOT INTENDED OR APPROVED FOR HUMAN, DIAGNOSTICS OR VETERINARY USE. Do not ingest, swallow or inhale. Do not get in eyes, on skin, or on clothing. Wash thoroughly after handling. For complete safety information see full Material Safety Data Sheet.

References

1.	Broedel, Jr., S. E. 2004. The I27RS8 Protein as a Reference for Force Extension Experiments. Athena Enzyme Systems™ Tech Bulletin #4, Athena Environmental Sciences, Inc.
2.	Czajkowsky, D. M. and Shao, Z. 1998. Submolecular resolution of single macromolecules with atomic force microscopy. FEBS Lett. 430:51-54.
3.	Carrion-Vazquez, M., Oberhauser, A. F., Fisher, T.K., Marszalek, P. E., Li, H., Fernandez, J. M. 2000. Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering. Prog. Biophys Mol. Biol. 74(1-2):63-91.
4.	Fisher, T. E., Oberhauser, A. F., Carrion-Vazques, M., Marszalek, P. E., and Fernandez, J. M. 1999. The study of protein mechanics with the atomic force microscope. TIBS 24:379-384.
5.	Carrion-Vazquez, M., Oberhauser, A. F., Fowler, S. B., Marszalek, P. E., Broedel, Jr., S. E., Clark, J., and Fernandez, J. M. 1999. Mechanical and chemical unfolding of a single protein: A comparison. Proc. Natl. Acad. Sci. USA. 96:3694-3699.
6.	Marko, J. F. and Siggia, E. D. 1995. Stretching DNA. Macromolecules 28:8759-8770.
7.	Oberhauser, A. F., Marszalek, P. E., Carrion-Vazques, M. and Fernandez, J. M. 1999. Single protein misfolding events captured by atomic force microscopy. Nature Struc. Biol. 6(11):1025-1028.
8.	Carrion-Vazquez, M., Marszalek, P. E., Oberhauser, A. F., and Fernandez, J. M. 1999. Atomic force microscopy captures length phenotypes in single proteins. Proc. Natl. Acad. Sci. USA 96:11288-11292.