WT-apoCA-tagRFP Biosensor Protein

WT-apoCA-tagRFP Biosensor Protein is a fluorescence-based biosensor with highest sensitivity for extracellular use.

Together with 4-(dimethylaminophenyl)benzoxazolyl sulfonamide, 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 an example of an 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 610 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.

Read plog post from Dr. Richard Thompson at Pokegama Technologies, Improving Zinc Ion Quantitation with Unique Biosensor Proteins and Buffers.

Catalog Number Product DataSheet Size AVAILABILITY Price Qty
KPG006
WT-apoCA-tagRFP Biosensor Protein
100ug In stock
Regular Price:$280.00
On Sale:
Specifications

Product Type: Protein
Name: wt-apoCA-tagRFP biosensor protein
Extinction Coefficient: ε555 nm = 100,001
Molecular Weight: 60,000 Da
Variant MPN: T-0005
Format: Solution in pH 7.5 buffer
Purity: >95%
Tested Applications: Extracellular measurement and imaging of free zinc ion at picomolar levels
Comments: Fluorescent-labeled zinc free apoprotein; fluorescent label excitation maximum 555 nm, preferred emission wavelength 612 nm
Storage: +4C
Shipped: Cold packs

Data
WtApo_Data
Comments
The sensor has extremely high affinity for Zn2+ (KD = 2.19 + 0.7pM), and Cu2+ (KD < 0.1 pM) as well as Hg2+; by comparison Cd2+, Co2+, and Ni2+ have affinities mostly in the nanomolar range. 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. Ca2+ and Mg2+ do not interfere at concentrations as high as 10 mM and 50 mM, respectively. For further details see our Resources on the Pokegama Technologies web site or our review: Bozym, et al., 2008 PMID 19152866.
References
  1. 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
  2. D. Wang, T. K. Hurst, R. B. Thompson, and C. A. Fierke ÒGenetically Encoded Ratiometric Biosensors to Measure Intracellular Exchangeable Zinc in Escherichia coliÓJ. Biomed. Opt. 16(8) 087011/1-11 (2011) [DOI: 10.1117/1.3613926. PMID: 21895338 PMCID: PMC3166341.
  3. R.A. Bozym, T. Hurst, N. Westerberg, A.V. Stoddard, C.A. Fierke, C.J. Frederickson, R.B. Thompson, ÒDetermination of zinc using carbonic anhydrase-based fluorescence biosensors,Ó in Methods in Enzymology: Fluorescence Spectroscopy Vol. 450 (L. Brand and M.L. Johnson, editors) New York: Elsevier, pp 279-301 (2008). PMID 19152866.
  4. R. A. Bozym, A. K. Stoddard, C. A. Fierke, and R. B. Thompson, ÒMeasuring picomolar exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor,Ó ACS Chemical Biology1(2) 103 Ð 111 (2006) PMID: 17163650.
  5. R. B. Thompson, M. L. Cramer, R. Bozym, and C. A. Fierke, ÒExcitation ratiometric fluorescent biosensor for zinc ion at picomolar levels,Ó J. Biomed. Optics7 (4), 555 - 506 (2002). PMID 1242112

If you publish research with this product, please let us know so we can cite your paper.

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