Malachite Green-NHS Ester (HPT1102)

Malachite Green-NHS Ester (MG-quenchTM ) is a technical grade fluorescent quenching labeling reagent. It is linked to primary amines on biomolecules such as peptides and oligonucleotides. Malachite green quenches fluorescence from molecules such as fluorescein derivatives. This water soluble molecule has wide applicability in labeling target molecules in biological systems. Malachite green can also serve as an alternative choice for the same role of Dabcyl, or Black hole quencher (BHQ) derivatives as a quencher for applications such as molecular beacons and probes.

Protocol: Peptide Conjugation

Suspend the peptide into a non-amine buffer at pH 8.3 (for example 10 mM Hepes, 150 mM NaCl, pH 8.3) to the highest concentration that the peptide remains soluble. Dissolve Malachite Green NHS Ester in DMSO at 100mg/ml, then immediately dispense into the peptide-buffer solution at a 100-1000X molar excess of Malachite Green NHS Ester relative to peptide. Assume a 30% purity of the technical grade Malachite-Green NHS Ester starting material, therefore multiply the required moles of Malachite-Green NHS Ester by x3 to correct the molar stoichiometry. Let the reaction proceed at RT with gentle shaking for 4 hrs. If you are conjugating malachite green to a protein in a cell lysate, add non-amine protease inhibitors and perform the reaction overnight at 4C with gentle shaking. The peptide-malachite green conjugate can be purified using gel filtration, or desalted in a spin column, by collecting the malachite green labeled peptide in the void volume. The malachite green-linked peptide should attain a green-blue coloration. The malachite-green labeled peptide can be followed at 616 nm in correspondence with the peptide bond UV absorbance peak. The final product is stored at -20C.


Protocol: Oligonucleotide Conjugation To Amino Modified Oligonucleotides

Malachite Green NHS Ester (30 mg, 90 micromole) is dissolved in 1.0 milliliter of methyl sulfoxide and 3 micromole amino modified oligonucleotide is dissolved in 800 microliter of 0.2 M sodium carbonate buffer (pH 8.5). The ester solution (400 microliter) is added to the amino-oligonucleotide solution. The mixture is left for 16 hour at 4C, after which it is chromatographed on a Sephadex G-25 column using de-ionized water/carbonate buffer (50/50) as eluent. The fraction with green-blue coloration with linked oligonucleotide is dialyzed against water to remove excess salts and unreacted reagents, and then freeze-dried. The final product is stored in the refrigerator until use.


Note about purity of Malachite Green NHS Ester:
The Malachite Green NHS ester is provided at technical grade. The purity of the Malachite Green NHS ester varies among lots, but impurities do not participate in the conjugation reaction with primary amine groups of target molecules. The labeled moiety of the target will only be Malachite Green. The customers should assume a 30% purity and use this value in their calculation for conjugation stoichiometry. If customers are interested in having highly pure NHS esters for conjugation that will lead to target Malachite Green labeled product, it is advised to use Leuco-Malachite Green NHS ester (FIVEphoton Part No. HPT1103), which has a purity greater than 97%. The conjugated Leuco-Malachite Green labeled target can be oxidized to Malachite Green by DDQ (2, 3-dichloro-5, 6-dicyano quinone) in situ as described in the protocol for the Leuco-Malachite Green NHS Ester reagent.