ERseeing (Endoplasmic Reticulum Green)

Product#: FDV-0038
$350.00
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ERseeing (Endoplasmic Reticulum Green)

Cat.No. FDV-0038
Size 10 nmol
Formulation: C37H28F2N2O4S
Molecular weight: 634.6g/mol
Solubility: Soluble in DMSO 
Ex/Em: 509 nm/524 nm
Storage -20 ºC

*FITC filter sets are available

Description


An irreversible ER staining reagent for live cell imaging. Compared to conventional fluorescently labeled Glibenclamide-based ER staining reagents, it has a smaller pharmacological effect on cell function and is irreversible, allowing observation even after medium replacement.

* This product is commercialized and sold by Funakoshi Co., Ltd. based on the research results of Professor Isao Hamachi and Professor Tomonori Tamura, Graduate School of Engineering, Kyoto University.
* This product is for research. It can only be used for research.

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.

ERseeing--F1.png

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.

How to use
 
General procedure of nucleus imaging
1. Prepare 0.1-1 uM ERseeing in serum-free medium.
NOTE: Highly recommend starting with 0.1 ?M ERseeing, higher concentrations such as 1 uM reagent may show non-specific staining. Empirically optimize and determine the concentration of ERseeing for your experiments.
 2. Remove culture medium and wash cells PBS several times
3. Add ERseeing-containing medium to cells.
4. Incubate cells at 37oC for over 15 min.
NOTE: Staining efficiency depends on incubation time. If you need to observe cells without washout step, 15 min staining is recommended. If you would like to observe stained cells after washout, 1-hour staining recommended. 
5. Wash cells with PBS or medium (Optional).
6. Observe cells under live condition or after fixation by 4% PFA and methanol.
 
ERseeing--F2.png

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.
ERseeing--F3.png
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).
ERseeing--F4.png
Comparison ERseeing between conventional dye
HeLa cells were treated with ERseeing or Glibenclamide-based 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.
ERseeing--F5.png

Reference

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