H94N-apoCA-tagRFP Biosensor Protein
Data:
Introduction:
This fluorescence-based biosensor has high sensitivity (K D = 0.22 nM) for measuring zinc intracellularly. Together with our 4-(dimethylaminophenyl)benzoxazolyl sulfonamide (Pokegama Cat. D-0005), measure cytoplasmic free zinc at concentrations below nanomolar (PMID 22430627, PMID 19152866, PMID: 17163650). It responds rapidly (see figure) to changes in free zinc at low levels which makes it useful for following rapid changes in free zinc concentration; see our TAT-H94N-apoCA-tagRFP (Pokegama Cat. no. T-0015) for a cell-penetrant variant potentially useful for measuring intracellular zinc transporter activity. It is one of our excitation ratiometric sensors, which makes accurate, reproducible calibration simple and minimizes most artifacts.
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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: H94N-apoCA-tagRFP biosensor protein
Fluorescent-labeled zinc free apoprotein; fluorescent label excitation maximum 555 nm, preferred emission wavelength 610 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 C.
Shipped: +4 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. Ca2+ at 10 mM and Mg2+ at 50 mM do not interfere. For further details see our review Bozym 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.