[살아있는 세포 내 소포체(세포질 망상 구조) 염색 시약/Endoplasmic Reticulum Green] ERseeing [FDV-0038]

ERseeing

Endoplasmic Reticulum Green

Cat.# FDV-0038 / Size: 10 nmol

 

ERseeing은 세포내 소기관인 ER을 염색하여 그 구조를 볼 수 있도록 도와줍니다.

ER의 다양한 생물학적 기능과 구조, 역할을 연구하는데 꼭 필요한 염색시약이라고 할 수 있습니다.

 

기존 ER 염색 시약으로 사용되는 Glibenclamide는 ATP-의존성 칼륨이온 통로 억제제로서 ER에 선택적으로 존재하는 sulphonylurea receptors 기능을 억제함으로써 관련 ER 연구에 있어 부정적인 영향을 미칩니다. 또한 가역적 억제제로서 배지를 교환하거나 세척하는 단계에서 염색 기능이 사라져 장시간 이미징 실험에 적합하지 않습니다.

 

ERseeing은 이러한 기존 ER 염색 시약의 문제점들을 극복하여 ER 기능을 유지하는 환경에서 ER 구조 이미징 및 연구 작업을 가능케 합니다. 

  

Product Background 

Endoplasmic reticulum (ER) is the largest organelle in the cell and has unique and dynamic tubular or sheet structures. ER plays essential roles in biosynthesis, precise folding and quality control of proteins and is a traffic origin of secreted pathway proteins including the Golgi apparatus, exocytosis, plasma membrane, and extracellular proteins. The major functions of ER are not only protein synthesis, but also carbohydrate metabolism, calcium storage, lipid metabolism, and lipid droplet synthesis. Visualization of ER structure in live cells is very important for the understanding of ER function and physiological significance of ER-resident proteins. 

 

The most conventional ER-staining dye is based on glibenclamide-fluorophore conjugate. Glibenclamide is known as a potent and specific inhibitor of the sulphonylurea receptors of ATP-sensitive K+ channels which are selectively localized on ER, glibenclamide-based ER dyes can visualize ER structures. However, its pharmacological activity negatively affects K+ channel functions in ER. In addition to the harmful influence of glibenclamide-based dyes for the cells, glibenclamide is a reversible inhibitor and can be washed out by wash step and medium change. Consequently, glibenclamide-based ER dyes can visualize only pharmacologically affected cells and not suitable for long-term imaging experiments. 

 

To overcome these problems, our ERseeing exhibit little effect on the ER functions pharmacologically and can visualize ER after washout or medium change. ERseeing has two units, a rhodol-type green fluorescent dye, and a thioester-type protein labeling group with rhodol-derivative having a high affinity to ER membrane. Right after addition of ERseeing to culture media, it can be accumulated into ER membranes. Protein labeling occurs with ERseeing non-specifically conjugates the rhodol fluorescent dyes onto ER-proteins by nucleophilic attack forming a covalent bond between ERseeing and ER-proteins resulting in a stable ER-rhodol label. ERseeing enables visualization of the ER structure even after washout or medium changes. This reagent is a powerful tool to monitor ER structures in live cells with little pharmacological effects.

 

Description 

Formulation: C37H28F2N2O4S 

Molecular weight: 634.6g/mol 

 Solubility: Soluble in DMSO 

Ex/Em: 509 nm/524 nm 

*FITC filter sets are available.

 

Application 

- Live cell ER imaging of cultured cells 

 

<NOTE> After staining live cells, cell fixation is compatible. However, this reagent does not stain ER specifically in fixed cells, staining step should be under live cell condition.

 

Reconstitution and Storage 

Reconstitution: stock solution recommended concentration 0.1 mM to 1 mM in 100% DMSO. 

Storage : Store powder at -20oC. After reconstitution in DMSO, aliquot and store at -20 °C. 

           Avoid repeated freeze-thaw cycles. Protect from light. 

 

Reference Data 

Absorption and fluorescent spectrum of ERseeing 

Excitation (blue) and fluorescent (red) spectrum. Exmax/Emmax = 509/524 nm. Commercial FITC filter sets are compatible.

 

 

ER specificity 

 

HeLa cells were stained with ERseeing (100 nM) and organelle markers, Glibencramide-type ER staining, lysosomal staining, mitochondrial staining, and Golgi apparatus staining. ERseeing was highly overlapped with conventional Glibencramide-type ER staining (Piason coefficiency >0.9) but not correlated with lysosome marker or mitochondria marker. Only a small portion of staining by ERseeing was overlapped with Golgi apparatus staining. It was considered that this is attributed to the vesicle transport of ERseeing or ERseeing labeling proteins from ER to Golgi apparatus. The ER-to-Golgi trafficking inhibitor decreased overlap between ERseeing-staining and the Golgi apparatus-staining (Detail information is described in Ref. 1).

 

Comparison ERseeing between conventional dye 

HeLa cells were treated with ERseeing or Glibenclamidebased dye for 15 min and observed without washout (Left). Both reagents show ER staining. After that, cells were washed by PBS, added fresh media containing 10% FBS and observed again. While the glibenclamide-based dye showed a very weak signal from the cells, ERseeing maintains a good signal from ER. ERseeing is suitable for long-term imaging after medium changes. 

 

* Research use only, not for human or animal therapeutic or diagnostic use !

 

References 

1. Fujisawa et al., J. Am. Chem. Soc., 140, 17060-17070 (2018) Chemical Profiling of the Endoplasmic Reticulum Proteome Using Designer Labeling Reagents.

 

코아사이언스 coresciences Funakoshi Inc. Japan 후나코시 한국 대리점 형광염색 시약 세포내 소기관 ER 소포체 관찰 이미징 이미지 구조 관찰 기능 연구