HBC  (Synonyms: HBC 530)

HBC (HBC 530) is a GFP fluorophore-like synthetic dye, with a structurally rigid electron acceptor and a strong electron donor. HBC has a low fluorescence background, and when combined with Pepper (RNA aptamer), HBC forms a tight complex and activates and emits bright fluorescence (K_d of ~3.5 nM). HBC emission peaks vary in different complexes and covers the spectrum from cyan to red. HBC can be used in the live cell imaging of RNA (Em/Ex = 530/485 nm)^[1].

Native polyacrylamide gel electrophoresis (PAGE) analysis
Native PAGE conditions included 8% PAGE gels (29:1 acrylamide:bisacrylamide) using TBE buffer.
The samples are run at 40 V for at least 2 h in pre-cooled buffer in an ice bath.
For staining, gels are incubated in buffer (40 mM HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl2) containing 10 μM HBC for 15 min.
Gels are then imaged to detect Pepper-HBC complexes. The stained gels are analyzed at 488/30 nm excitation and 535/30 nm emission.
Next, counterstaining with 1× SYBR Gold is performed for 15 min followed by gel imaging to detect all RNA species in each lane.
SYBR Gold-stained bands are imaged using ultraviolet excitation (302 nm) and 590/30 nm emission.

Mammalian cell flow cytometry
293T/17 cells are transfected with the corresponding plasmids. Empty plasmid without expression of aptamer is used as the control. Untransfected cells are used as a negative control.
After 30 h, the cells are digested and resuspended in the 4% FBS/1× PBS solution containing 0.2 μM HBC and 5 mM MgSO4.
The fluorescence is analyzed using a cytometer with two channels: Ex=488/8 nm, Em=525/40 nm and Ex=561/10 nm, Em=610/20 nm.

To study the correlation between mRNA and protein, cells are transfected with plasmids expressing different mRNAs tagged with 4Pepper and cultured for 24 h.
The cells are digested and resuspended in the 4% FBS/1× PBS solution containing 0.2 μM HBC and 5 mM MgSO4.
Cells expressing SNAP-4Pepper are pre-treated with 0.2 μM SNAP-Cell 647-SiR (NEB).
Ex=405/10 nm, Em=450/45 nm, Ex=488/8 nm, Em=525/40 nm, Ex=561/10 nm, Em=610/20 nm and Ex=638 nm, Em=660/20 nm channels are used for blue fluorescent protein, Pepper530, mCherry and SNAP-SiR, respectively.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

303.36

C₁₉H₁₇N₃O

156840-13-0

Solid

Orange to red

530

485

N#CC1=CC=C(/C(C#N)=C\C2=CC=C(N(CCO)C)C=C2)C=C1

Room temperature in continental US; may vary elsewhere.

4°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Chen X, et al. Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat Biotechnol. 2019;37(11):1287-1293.  [Content Brief]