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Mobile Zinc-Responsive Protein Labeling Reagent Zinc-Stamp™ | Funakoshi

Date：November 20 2020Web Page No：80849

Funakoshi Co.,Ltd.

Zinc-Stamp™ is a chemically labeling reagent that is activated by mobile zinc ion (Zn2+) and thereby covalently attaches a fluorescein tag to proximal proteins. After treatment of cells with Zinc-Stamp™ followed by cell fixation, existence and localization of Zn2+ can be analyzed by fluorescent microscopy. In addition, Zinc-Stamp™ can be used to analyze and identify proteins involved in Zn2+ dynamics and transport.


Notice: Product Name Changes

This product has been renamed as follows:
※ There is no change in specifications, catalog no. and size.

  • Old name : Zin-Pro Capture <Mobile Zinc-Responsive Protein Labeling Reagent>
  • New name : Zinc-Stamp™ <Mobile Zinc-Responsive Protein Labeling Reagent>

Product image of Zinc-Stamp&trade

Product image of Zinc-Stamp™

Importance of mobile zinc ion (Zn2+) analysis

Zinc is an essential element for living organisms and exist in high concentration in cells. It is well known that zinc plays important roles in protein folding and protein functions such as enzyme activities. It is estimated that 10% of total proteins are zinc-binding proteins. In normal state of cells, zinc ion (Zn2+) is stably stored in zinc-binding proteins such as metallothionein and thereby intracellular mobile Zn2+ is kept at low concentrations. On the other hand, several functions of mobile Zn2+ including function as a signal transducing factor via moderate binding on surface of proteins have been reported. Furthermore, close relationships between excess mobile Zn2+ and several diseases such as cerebral ischemia, brain injury, epilepsy, and Alzheimer's diseases are indicated. Therefore, homeostasis of mobile Zn2+ has attracted much attention in broad field of life science.

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What is Zinc-Stamp™?

Zinc-Stamp™ is a chemically labeling reagent that is activated by mobile zinc ion (Zn2+) and thereby covalently attach a fluorescein tag to proximal proteins. Zinc-Stamp™ (the name in original paper : ☞ AIZin-2) was developed by professor Itaru Hamachi and co-workers in Kyoto University. Zinc-Stamp™ is attracting attention as a useful chemical tool for analyzing the intracellular behavior and dynamics of mobile zinc in protein level.

Zinc-Stamp™(AIZin-2)

Zinc-Stamp™ (☞ AIZin-2)

The principle of protein labeling

Zinc-Stamp™ has the structure that consist of Zn2+-chelator containing acyl imidazole and fluorescein. The initial state of Zinc-Stamp™ is inactive to protein labeling. On the other hand, once Zn2+ is chelated by dipicorylamine and acyl imidazole of Zinc-Stamp™, the acyl imidazole is activated and fluorescein labeling reaction to proximal proteins occurs. This molecular design is enabling protein labeling in response to the existence of Zn2+ in the surrounding environment.

Principle of Zinc-Stamp™

Principle of zinc-responsive protein labeling reagent

Cell imaging with Zinc-Stamp™

Zinc-Stamp™ is activated by Zn2+ and labels a fluorescein to proximal proteins in cellular region where mobile Zn2+ is highly concentrated.

Cell imaging with Zinc-Stamp™

Zinc-Stamp™ can visualize the amount and the local site of Zn2+ after fixation of the cells, while it cannot observe Zn2+ localization in living cells before fixation because of the overwhelmed fluorescence derived from the excess unreacted Zinc-Stamp™.

Living Cells   Fixed Cells
TPEN (−) TPEN (+) TPEN (−) TPEN (+)
Living Cells TPEN (−) Living Cells TPEN (+)

Fixation



Unreacted fluorescent dyes are removed by fixation

 Fixed Cells TPEN (−) Fixed Cells TPEN (+)
  • Green:Zinc-Stamp™ 
  • Blue:Nucleus

Fluorescent imaging of human kidney cancer 786-O cells with Zinc-Stamp™ in the condition of Zn2+ depletion by Zn2+-chelator TPEN

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Features

  • Specific for Zn2+. Not reactive with Mn2+, Fe2+, Fe3+, Na+, K+, Ca2+, and Mg2+
  • High S/N ratio
  • Low cytotoxicity
  • High cell membrane permeability - Just add Zinc-Stamp™ to culture medium
  • Labeled proteins can be purified by anti-fluorescein antibody
  • Works with various applications (☞ see below)
  • Formulation : C46H40N6O11
  • Molecular weight : 852.85 g/mol
  • Solubility : Soluble in DMSO
  • Ex/Em : 495/515 nm (compartible with general FITC filter)

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Application data

Application classification


  Treatment of biological sample with Zinc-Stamp™  
   
  Protein labeling with Zinc-Stamp™  
       
         
Cell fixation   Preparation of cell lysate
               
Immunostaining   SDS-PAGE   Immunoprecipitation with fluorescein-antibody
                 
         
Fluorescent microscopy   Analysis of proteins
with fluorescent signal		   Western blot with antibody
for protein of interest 		  Proteomics analysis
with MS
🢃   🢃   🢆   🢇
☞ Application① ☞ Application② ☞ Application③

Application① Imaging of dynamics and localization of mobile Zn2+ in cells

The localization of proteins labeled with Zinc-Stamp™ can be visualized by fixing cells. Co-staining with antibodies or organelle-markers can be performed, which enable to analyze the relationships between mobile Zn2+ dynamics and protein of interest, such as Zn2+ transport proteins.

SNOC 10 min   SNOC 2 hour

SNOC 10 min
SNOC 2 hour

Cells were treated with S-nitrosocysteine (SNOC; NO-generating reagent) and labeled by Zinc-Stamp™. Cells were fixed by methanol and analyzed with confocal fluorescent microscopy. Fluorescent responses were observed throughout the cell after 10 minutes of oxidative stress, whereas vesicle-like fluorescent signals were observed after 2 hours of oxidative stress. This indicated that mobile Zn2+ was transported to vesicles to suppress toxicity.

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Application② Analysis of total labeled proteins by SDS-PAGE

Application② : Analysis of total labeled proteins

C6 glioma cells were treated with 200 µM SNOC, a NO generator, for 0, 15 and 180 min. After NO-stimulation, cells were incubated with 1 µM Zinc-Stamp™ for 10 min in the absence or presence of 400 µM TPEN, a potent Zn-chelator. Cells were lysed by SDS-sample buffer and proteins were separated in SDS-PAGE and detected by fluorescent imager for labeled proteins or CBB staining for total proteins. Band pattern of labeled proteins was dramatically changed by SNOC treatment and fluorescent signals were suppressed in the presence of TPEN.

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Application③ Analysis of labeled proteins by WB & MS

C6 glioma cells were treated with 200 µM SNOC for 0, 10, and 180 min and incubated with 10 µM Zinc-Stamp™ for 10 min. After lysis of cells, labeled proteins were immunopurified by fluorescein antibody (anti-FL). Analysis were done in ☞ Western Blot(A) and ☞ Mass Spectrometer(B).


(A) Analysis of individual proteins with western blot

A: Analysis of individual proteins with western blot

Isolated proteins by anti-FL were separated by SDS-PAGE and detected by western blotting with antibodies of interest (IP-blot). In this case, mitochondrial protein (citrate synthetase), ER protein (calnexin) and Golgi apparatus protein (GRP94) were analyzed. These results indicate mobile Zn2+ was released from metallothionein by NO treatment, transiently accumulated in mitochondria within 10 min and finally transported to ER and Golgi apparatus.

(B) Proteomics analysis with MS

Isolated proteins by anti-FL were separated by SDS-PAGE and digested by trypsin. Then, each sample was labeled with a stable isotope by the TMT method. Comparative analysis was performed using LC-MS/MS with combinations of NO treatment of 0 minutes/10 minutes and 10 minutes/180 minutes. The change in the amount of proteins labeled with Zinc-Stamp™ between samples could be comprehensively analyzed.

B: Proteomics analysis with MS (NO 0 min vs 10 min)   B: Proteomics analysis with MS (NO 10 min vs 180 min)
NO 0 min vs 10 min NO 10 min vs 180 min

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Cited by

  • Miki, T., et al., "A conditional proteomics approach to identify proteins involved in zinc homeostasis.", Nat. Methods, 13(11), 931～937 (2016). [PMID : 27617391]

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Product Information

[Date : April 25 2025 00:06]

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

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

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

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

[Date : April 25 2025 00:06]

Zinc-Stamp, Mobile Zinc-Responsive Protein Labeling Reagent

DatasheetThis may not be the latest data sheet.


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

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

Zinc-Stamp, Mobile Zinc-Responsive Protein Labeling Reagent

DatasheetThis may not be the latest data sheet.


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

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

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