E117A-apoCA-tagRFP Biosensor Protein
Data:
Introduction:
This fluorescence-based biosensor has high sensitivity (KD = 0.04 nM) for measuring zinc, but responds much faster to changes in free zinc at low levels than the wild type carbonic anhydrase-based sensors (see figure) to changes in free zinc at low levels. Together with our 4-(dimethylaminophenyl)benzoxazolyl sulfonamide (Cat. D-0005), it enables measurement of free zinc in aqueous media near neutrality at concentrations below nanomolar (PMID 22430627, PMID 19152866, PMID: 17163650). 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; soluble in water at pH> 6
Product Type: Fluorescence-based metal ion biosensor
Name: E117A-apoCA-tagRFP biosensor protein
Fluorescent-labeled zinc-free apoprotein; fluorescent protein label excitation maximum 555 nm, preferred emission wavelength 584 nm
Description:
Molecular Weight : approx. 60,000
Format: solution in pH 7.5 buffer
Purity: >95%
Tested Application: Measuring free zinc ion concentrations
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 ≈ 0.04 nM), as well as Cu2+, Hg2+, Cd2+, Co2+, and Ni2+, with affinities mostly in the picomolar to 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 Resource pages on the Pokegama Technologies web site and the review: Bozym, et al., (2008) PMID 19152866.
References:
T. K. Hurst, D. Wang, R.B. Thompson, C.A. Fierke, “Carbonic anhydrase II-based metal ion sensing: Advances and new perspectives,” Biochimica et Biophysica Acta (Proteins and Proteomics)1804, 393-403 (2010). PMID: 19818877; NIHMSID 157045.
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