BHQ-3 Carboxylic Acid, Succinimidyl Ester
BHQ-3 Carboxylic Acid, Succinimidyl Ester
Key featuresShow Hide
- Used to conjugate a non-fluorescent quencher to an amine functionality of a biomolecule.
- Maximal absorption in the 620 to 730 nm range.
Cannot add to favourites
An option must be selected in order to add to favourites
Since their introduction to the DNA marketplace in 2000, the Black Hole Quenchers have become THE gold-standard quencher of choice used in qPCR probes and other FRET applications. When fluorogenic dual-labeled probes were first introduced, quenchers were often a second reporter dye, typically TAMRA. The sensitivity of these probes, such as FAM-TAMRA, is limited because the fluorescence from TAMRA can leak into the channel meant to detect FAM fluorescence. Dark quenchers, which are dyes with no native fluorescence, offer a solution to this problem. The BHQ dyes are true dark quenchers with no native emission due to their polyaromatic-azo backbone. Substituting electron-donating and withdrawing groups on the aromatic rings produces a complete series of quenchers with broad absorption curves that span the visible spectrum into the near IR region. BHQ dyes work through a combination of FRET and static quenching to enable researchers to avoid the residual background signal common to fluorescing quenchers such as TAMRA, or low signal to noise ratio. These quenchers can be paired with all common reporter dyes to construct efficiently quenched qPCR probes for multiplexing assays. In addition to quenching by FRET, BHQ dyes have also been shown to efficiently quench fluorescence through static quenching via formation of a ground state complex with the reporter dye. BHQ quenchers have broad absorption spectra and can be paired with reporter dyes that emit in the following ranges: BHQ-0: 430-520 nm BHQ-1: 480-580 nm BHQ-2: 560-670 nm BHQ-3: 620-730 nm BHQ-10: 480-550 nm Water Soluble (WS) LGC, Biosearch Technologies offers all Black Hole Quencher products available for 3’, internal and 5’ modification of oligonucleotides with a variety of options. BHQ-1 and BHQ-2 are the more popular, either as the 5'-Phosphoramidites, the dT-Phosphoramidites or the 3'-CPGs. Only considering the excitation and emission values suggests Cy™5/Cyanine-5 and Quasar 670 require BHQ-3 for efficient quenching, however BHQ-2 is recommended because it is less susceptible to degradation. BHQ-1 is typically used to quench in the range 480 - 580 nm and can be used in conjunction with the commonly used fluorophores; e.g. FAM, TET, JOE and HEX. BHQ-2 is used to quench in the range 550 – 650 nm and is most effective in quenching fluorophores such as TAMRA, ROX, Cyanine-3, Cy3, Cy3.5™ and Red 640. We also offer Black Hole Quenchers for labelling peptides. All of our BHQ dyes are available as carboxylic acid or succinimidyl esters. Our BHQ-1 and BHQ-2 dyes are available as FMOC lysine conjugates. Our water soluble BHQ-10 is available as a carboxylic acid or succinimidyl ester.
- Formula: C37H38F6N7O4P
- Molecular Weight: 789.71
- Appearance: dark purple solid
- Absorption Maximum (Lambda Max): 615
- Extinction Coefficient at Lambda max: 40700
- Extinction Coefficient at 260 nm: 13000
Spectral properties measured in phosphate-buffered saline (PBS) pH 7.3.
- Labeling peptides with dyes is an art that requires considerable trial and error even with experience, and each different peptide demands a unique optimization. For additional information you may review the reference publications, particularly the procedures listed in their Supplemental Information. Given the difficulties in synthesizing and labeling peptides, you may also want to consider having them manufactured for you with the dye label(s) pre-attached, by companies that specialize in the task.Please note: The BHQ Succinimidyl ester products are intended for conjugation to primary amines such as the N-terminus and if the sequence contains residues like Lysine that present an additional primary amine then you may get multiple incorporations of the BHQ onto each peptide molecule.DIPCDI + HOAt Procedure for Dye Carboxylate Addition to Peptide on Solid Phase:All products bearing BHQ groups are susceptible to reduction when using DTT or other reducing agents.DIPCDI stock solution. 1 mL DIPCDI (di-isopropylcarbodiimide) + 9 mL DMF (HPLC quality).HOAt (1,2,3-Triazolo-[4,5-b]pyridin-3-ol, 1-hydroxyl-7-azabenzotriazole, TCI America T1673), 10 mg solid per 10 mg dye.Procedure: The peptide resin, 20mg, is placed in a syringe reactor (2ml polypro with coarse PE end frit), and the resin swollen in DMF. The dye (10mg) and HOAt (10mg) are placed in a screw capped 1.5 ml vial are treated with 110 L of freshly prepared DIPCDI solution, the vial sealed, and vortexed vigorously for 2 minutes. The dye solution is then diluted with DMF (200 L), mixed and admitted to the reactor. This is covered with foil and shaken overnight. The resin is washed 5x with DMF, treated 1x1 min with 30% piperidine in DMF, washed further with DMF (at least 5x) until no color in washes, washed 5x methanol, and all solvent expelled from syringe. Cleavage reagent (95%TFA/5% H20 made up under argon, 1.5 mL) is admitted to the syringe, which is shaken for 90 minutes. The TFA solution is then expelled into a 20mL glass scintillation vial and evaporated. The residue is dried in vacuo over KOH pellets. The product is analyzed by standard C-18 analytical HPLC in TFA/acetonitrile buffers with a PDA detector.Hazards: This procedure, as do all peptide synthesis protocols, involves handling many dangerous, toxic, corrosive, irritant and poisonous chemicals. It should only be performed by a very experienced chemist in a laboratory fume hood with all possible caution. All waste should be disposed of according to safe laboratory procedures. Particular note should be made of the fact that HOAt is rated as being potentially explosive; it should be handled with care avoiding shock, heat or static electricity. Biosearch Technologies, Inc. assumes no responsibility for the safety of any part of this procedure, which has been supplied strictly for informational purposes only.Polster, B. M., Arze, R., Lyttle, M. H., Nicholls, D. G., & Hudson, D. (2007). “Solid Phase Synthesis of Dual Labeled Peptides: Development of Cell Permeable Calpain Specific Substrates. International Journal of Peptide Research and Therapeutics”, 13(1), 83-91.Lee, Seulki, et al. "Dark quenched matrix metalloproteinase fluorogenic probe for imaging osteoarthritis development in vivo." Bioconjugate chemistry 19.9 (2008): 1743-1747.Stefflova, K., Chen, J., Marotta, D., Li, H., & Zheng, G. (2006). “Photodynamic therapy agent with a built-in apoptosis sensor for evaluating its own therapeutic outcome in situ” Journal of medicinal chemistry, 49(13), 3850-3856.
Storage and handling:
- Shipping conditions: Cold
- Storage conditions: -15 to -30 °C