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Mobile Zinc-Responsive Protein Labeling Reagent Zin-Pro Capture | Funakoshi

Date:November 20 2020Web Page No:80849

Funakoshi Co.,Ltd.

Zin-Pro Capture
- A protein labeling reagent in the presence of mobile zinc in the cell -

Zin-Pro Capture is a chemically labeling reagent of a fluorescein tag to proximal proteins only in the presence of mobile Zn(II) ion. Zin-Pro Capture is useful for analyses and assays of protein which relates to transport kinetics of mobile zinc in the cells.

So far, to prove intracellular behavior of Zinc ion, imaging probes for metal Zinc has been used.
Such probes can only detect the location of Zinc in the cell, and it was difficult to find which proteins are related to transportation of Zinc. This is because current Zinc imaging probe is compatible with live cell imaging but not optimized for immunochemistry, as such probes are not suited for fixation.

Zin-Pro Capture is a labeling reagent which overcomes such disadvantage.

This product is commercialized by the research result of Professor Hamachi, at Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University.
Miki et al., Nat. Methods 13, 931-937 (2016)
* In this paper, Zin-Pro Capture is called as AIZin-2.

Background

Zn2+ is tightly bound to proteins and plays essential roles in precise protein folding, and enzymatic function of metalloproteins as cofactor. A simulation suggests about 10% of proteins are estimated as Zn2+-binding proteins1). Mobile Zn2+, which is free form or loosely bound to protein surface, is the second class of zinc population and act as a signaling molecule. Recent evidences show an abnormal increase in mobile Zn2+ concentration induce pathological events including neuronal degeneration etc. Although fluorescent probes for real time monitoring of mobile Zn2+ in live cells have been developed, these probes could not reach comprehensive characterization of Zn2+ distribution mechanism and how Zn2+ mobiles in cells. To approach zinc homeostasis in cells and to identify proteins involved in zinc distribution on the molecular level, chemical reagents which can be applied for protein experiments are desirable. Zin-Pro Capture is a chemically labeling reagent of a fluorescein tag to proximal proteins only in the presence of mobile Zn2+ ion (Figure 1). Zin-Pro Capture has high selectivity for Zn2+ ion, not showing labeling activity with the other abundant metal ions such as Mn2+, Fe2+/Fe3+, Ca2+ and Mg2+. Its Zn2+-responsive labeling activity enable to monitor mobile Zn2+ species in the cell on the molecular basis. After labeling Zn-proximal proteins with fluorescein tag in living cells by Zin-Pro Capture, cells are lysed and the labeled proteins are subsequently enriched by immunoprecipitation with anti-fluorescein antibody. The labeled and purified proteins are characterized by western blotting with antibodies against interest protein or comprehensively identified by mass analysis. Observation of intracellular localization of labeled proteins by Zin-Pro Capture is also available in the fixed cells. Miki el al. showed that mobile Zn2+ in C6 glioma cells was dramatically released to cytosol from metal binding proteins such as metallothionein by oxidative stress and subsequently accumulated in vesicles2). This study revealed with Zin-Pro Capture (AIZin2 in the paper2)) that oxidative stress induced Zn-rich vesicles in glioma cells were identified as COPII, COPI and ER-Golgi intermediate compartment (ERGIC) between ER and Golgi appratus2-3).

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Principle

In the absence of Zn(II) ion, protein labeling activity of Zin-Pro Capture is poor. On the other hand, in the presence of Zn(II),  Zin-Pro Capture is activated by mobile Zn(II) and shows protein labeling activity of fluorescent tag to proximal proteins.

In the absence of Zn(II) ion, protein labeling activity of Zin-Pro Capture is poor. On the other hand, in the presence of Zn(II), Zin-Pro Capture is activated by mobile Zn(II) and shows protein labeling activity of fluorescent tag to proximal proteins.

Principle

Chemical labeling activity of Zin-Pro Capture is dependent on the amount of Zn(II) ion in the cell. Highly concentrated Zn(II) increases Zin-Pro Capture activity and proteins located on Zn(II) rich compartment are densely labeled.
On the other hand, in the Zn(II) poor region, Zin-Pro Capture shows low efficiency and little proteins are labeled.
The labeled proteins can be purified by immunoprecipitation with anti-fluorescein antibody and applied for western blotting and mass spectrometry etc and also observed by microscopy in the fixed cells.

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Features

  • Specific for Zn (II). Not reactive for Mn(II), Fe(II), Fe(III), Na(I), K(I). Ca(II), and Mg(II)
  • High S/N ratio
  • Low cytotoxicity
  • High cell membrane permeability - Just add Zin-Pro Capture to culture medium
  • Labeled protein can be purified by Anti-fluorescein antibody
  • Compatible with immunochemistry after fixation of cells
  • Compatible with other assays, such as SDS-PAGE, Western Blotting, and MS spectrometry (LC-MS/MS)
  • IUPAC name: 5-((2-(2-((4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-imidazole-1-carbonyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl diacetate
  • Formulation : C46H40N6O11
  • Molecular weight : 852.85 g/mol
  • Solubility : Soluble in DMSO
  • Ex/Em:495/515 nm (Compartible with general filter for FITC)

* Zin-Pro Capture is not optimized for live cell imaging.

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Related Research

  • Zinc homeostasis
  • Neurodegenerative disorder, related to excessive/deficient of Zinc
Application Conventional Zinc-imaging reagents Zin-Pro Capture
Analyzing total proteins related to zinc homeostasis by IP, MS and WB ×
Monitoring zinc-accumulation sites ×
Identifying mobile zinc transport proteins ×
Finding mobile zinc regulation ×
Live cell imaging ×
Seeing zinc ion itself ×

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Data

Fig.1 Localization of labeled protein in the cells

Fig.1 Localization of labeled protein in the cells
C6 glioma cells were treated by S-nitrosocysteine (SNOC ; NO producing reagent), and labeled by Zin-Pro Capture.
Cells were fixed by methanol.

Fig. 2 Analysis of labeled proteins in western blot and Mass Spectrometer

Fig. 2 Analysis of labeled proteins in western blot and Mass Spectrometer
C6 glioma cells were treated by S-nitrosocysteine 200 μM and purified by anti-fluorescein antibody (anti-FL). Analysis were done in western blot (A) and Mass Spectrometer (B).
(A) Immunoprecipitated proteins were detected in Western Blot. Strong band means the protein is located in the Zinc-rich region. Citrate synthetase (mitochondrial protein) was detected at 10 minutes after treatment, and Calnexin and GRP 94 (ER proteins) were detected at 180 minutes after treatment.
This concludes that mobile zinc were released from metallothionein in cytoplasm, and transported to ER by way of Mitochondria.
(B) Assay by Mass Spectrometer 0, 10, 180 minutes samples were labeled with stable isotope by TMT method, and analyzed by LC-MS/MS.

Fig.5

Zin-Pro Capture reveals mobile zinc under oxidative stress
- transiently transported into mitochondria
- accumulated in ER and ER-Golgi intermediate compartment (ERGIC).

in vitro ischemia model
Cell : C6 glioma
Stimuli : Nitric oxide-based oxidative stress

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Reference

1) Andrein et al., J. Proteome Res., 5, 196–201 (2006).
2) Miki et al., Nat. Methods., 13, 931-937 (2016)
3) Motiwala and Martin, Nat. Methods., 13, 917-918 (2016)

[Date : January 26 2021 23:29]

Detail Product Name Product Code Supplier Size Price
Zin-Pro Capture <Mobile Zinc-Responsive Protein Labeling Reagent>
DatasheetThis may not be the latest data sheet.
FDV-0013A FNAFunakoshi Co.,Ltd. 25 µg $400

Description Zin-Pro Capture is a chemically labeling reagent of a fluorescein tag to proximal proteins only in the presence of mobile Zn(II) ion. Zin-Pro Capture is useful for analyses and assays of protein which relates to transport kinetics of mobile zinc in the cells.
Storage -20°C CAS
Link

Zin-Pro Capture <Mobile Zinc-Responsive Protein Labeling Reagent>
DatasheetThis may not be the latest data sheet.
FDV-0013B FNAFunakoshi Co.,Ltd. 3x25 µg $1,000

Description Zin-Pro Capture is a chemically labeling reagent of a fluorescein tag to proximal proteins only in the presence of mobile Zn(II) ion. Zin-Pro Capture is useful for analyses and assays of protein which relates to transport kinetics of mobile zinc in the cells.
Storage -20°C CAS
Link

[Date : January 26 2021 23:29]

Zin-Pro Capture <Mobile Zinc-Responsive Protein Labeling Reagent>


  • Product Code: FDV-0013A
  • Supplier: FNA
  • Size: 25µg
  • Price: $400

Description Zin-Pro Capture is a chemically labeling reagent of a fluorescein tag to proximal proteins only in the presence of mobile Zn(II) ion. Zin-Pro Capture is useful for analyses and assays of protein which relates to transport kinetics of mobile zinc in the cells.
Storage -20°C CAS
Link

Zin-Pro Capture <Mobile Zinc-Responsive Protein Labeling Reagent>


  • Product Code: FDV-0013B
  • Supplier: FNA
  • Size: 3x25µg
  • Price: $1,000

Description Zin-Pro Capture is a chemically labeling reagent of a fluorescein tag to proximal proteins only in the presence of mobile Zn(II) ion. Zin-Pro Capture is useful for analyses and assays of protein which relates to transport kinetics of mobile zinc in the cells.
Storage -20°C CAS
Link

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CONTACT

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