BHQ-10 Carboxylic Acid, Succinimidyl Ester
BHQ-10 Carboxylic Acid, Succinimidyl Ester
Key featuresShow Hide
- Used to conjugate a non-fluorescent quencher to an amine functionality of a biomolecule.
- Maximal absorption around 516 nm.
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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: C27H24N6Na2O10S2
- Molecular Weight: 702.63
- Appearance: dark red solid
- Absorption Maximum (Lambda Max): 516
- Extinction Coefficient at Lambda max: 28700
Spectral properties measured in phosphate-buffered saline (PBS) pH 7.0.
- The absorption maximum of BHQ-10 can be sensitive to changes in pH.The extinction coefficient at 516 nm is 28,700/M/cm (PBS, pH, 7.0); 30,400/M/cm (bicarbonate buffer, pH 8.5)The extinction coefficient at 260 nm is 7,900/M/cm (PBS, pH, 7.0); 8320/M/cm (bicarbonate buffer, pH 8.5)The extinction coefficient at 280 nm is 8,200/M/cm (PBS, pH, 7.0); 8680/M/cm (bicarbonate buffer, pH 8.5)Product Solubility:This product is soluble in DMF and DMSO. It is also soluble in aqueous media but once dissolved the succinimidyl ester reactive group will hydrolyze to give the carboxylic acid which is non-reactive.Method for conjugation of BHQ10-OSu to proteins and antibodies:• BHQ-10 OSu is NOT COMPATIBLE with tris buffers• Use carbonate-bicarbonate, borate or phosphate buffer, pH 7.5 to 9.0, for conjugation of BHQ-10 OSu to proteins. • Prepare a stock solution of BHQ 10-OSu in DMF or DMSO and immediately add to the aqueous solution of protein (1-2 mg/mL) such that the organic solvent is no more than 10% of the final volume. Alternatively, add the solid BHQ-10 OSu to the protein solution and vortex or sonicate to ensure complete dissolution.• Separate the labeled protein from excess dye by size-exclusion chromatography, e.g. Sephadex G50, or by dialysis.Do not store stock solutions of this product for longer than two days.Product Stability:Reducing agents that are commonly used to reduce dithiol bonds in proteins, such as DTT and TCEP, will also reduce BHQ-10 thereby causing a loss of color and of quenching efficiency.Conjugation of BHQ-10 OSu to substrates in aqueous media:Amine reactive reagents such as succinimide esters react with non-protonated aliphatic amino groups, including the amine terminus of proteins and the epsilon-amino group of lysines. To maintain the amine groups in the non-protonated form, the conjugation reaction must take place in a buffer with slightly basic pH. More specific labeling of the amine terminus may be achieved by using a buffer closer to neutral pH, as the pKa of the terminal amino group is lower than that of the lysine epsilon amino groups.Buffers, such as Tris, that contain primary amines should not be used with this product. Instead borate, phosphate or carbonate buffers should be used. For best results with this product, we recommend using the buffers specified in the protocol below. The procedure may be scaled up or down, maintaining the same molar ratios of reagents. It is important to consider that the number and surface positions of the amino groups will vary greatly among proteins. The reactivity of the amino groups on proteins will vary according to the structure of the protein. If possible, we recommend experimentation with different molar ratios of reactive dye to protein in order to determine the protocol that yields the desired level of incorporation into the protein.
Storage and handling:
- Shipping conditions: Cold
- Storage conditions: -15 to -30 °C
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