wt-apoCA-tagRFP Biosensor Protein
Data:
Introduction:
This is our prototype fluorescence-based biosensor with highest sensitivity for extracellular use. Together with our 4-(dimethylaminophenyl)benzoxazolyl sulfonamide (Cat. D-0005), one can measure free zinc in aqueous solution at concentrations in the picomolar range (PMID 22430627, PMID 19152866, PMID: 17163650). The protein is a fusion protein of wild type apocarbonic anhydrase together with tagRFP. It is one of our excitation ratiometric sensors, which makes accurate, reproducible calibration simple and minimizes most artifacts: the principle is illustrated below. When zinc is not bound, the 4-(dimethylaminophenyl)benzoxazolyl sulfonamide does not bind and emission at 617 nm (or other suitable wavelength) is weak when excited at 365 nm. However, when zinc binds to the carbonic anhydrase active site, the sulfonamide can now bind with a substantial increase in quantum yield, and efficiently transfers its energy to the attached tagRFP, which then emits in the red. Directly exciting the tagRFP at 555 nm enables one to normalize the UV-excited Zn-bound form against the total tagRFP as an excitation ratio.
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Specifications: mol wt 60,000 ε555 nm = 100,000;
Product Type: Fluorescent-labeled zinc-free apoprotein
Name: wt-apoCA-tagRFP biosensor protein
Fluorescent-labeled zinc free apoprotein; fluorescent label excitation maximum 555 nm, preferred emission wavelength 612 nm
Description:
Molecular Weight : approx. 60,000 Daltons
Format: solution in pH 7.5 buffer
Purity: 95+%
Tested Application: Extracellular measurement and imaging of free zinc ion at picomolar levels
Solubility: aqueous buffer, pH >6.0
Storage: 4 degrees C.
Shipped: 4 degrees C.
Provider:
Pokegama Technologies, Inc. (KeraFast Partner)
Comments:
The sensor has extremely high affinity for Zn2+ (KD ≈ pM), and Cu2+ (KD < 0.1 pM) as well as Hg2+, Cd2+, Co2+, and Ni2+, with affinities mostly in the nanomolar range. Thus the sensor should only be dissolved in metal-free buffers (or our metal ion buffers with known free zinc concentration), and should not touch metal or glass; most plastic (including natural pipette tips) is satisfactory in this regard. For further details see our Resources on the Pokegama Technologies web site or our review: Bozym, et al., 2008 PMID 19152866.
References:
B. J. McCranor, R. A. Bozym, M. Vitolo, C. A. Fierke, L. Bambrick, B. Polster, G. Fiskum, and R. B. Thompson, "Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion" Journal of Bioenergetics and Biomembranes 44(2) 253 - 263 (2012). DOI: 10.1007/s10863-012-9427-2 PMID 22430627 NIHMS382081
D. Wang, T. K. Hurst, R. B. Thompson, and C. A. Fierke “Genetically Encoded Ratiometric Biosensors to Measure Intracellular Exchangeable Zinc in Escherichia coli”J. Biomed. Opt. 16(8) 087011/1-11 (2011) [DOI: 10.1117/1.3613926. PMID: 21895338 PMCID: PMC3166341 .
R.A. Bozym, T. Hurst, N. Westerberg, A.V. Stoddard, C.A. Fierke, C.J. Frederickson, R.B. Thompson, “Determination of zinc using carbonic anhydrase-based fluorescence biosensors,” in Methods in Enzymology: Fluorescence Spectroscopy Vol. 450 (L. Brand and M.L. Johnson, editors) New York: Elsevier, pp 279-301 (2008). PMID 19152866.
R. A. Bozym, A. K. Stoddard, C. A. Fierke, and R. B. Thompson, “Measuring picomolar exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor,” ACS Chemical Biology1(2) 103 – 111 (2006) PMID: 17163650.
R. B. Thompson, M. L. Cramer, R. Bozym, and C. A. Fierke, “Excitation ratiometric fluorescent biosensor for zinc ion at picomolar levels,” J. Biomed. Optics7 (4), 555 - 506 (2002). PMID 1242112