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Reactive Tag Viscosity Probes

Ursa BioScience uniquely offers several of our Viscosity probes with reactive groups / tags for attachment to thiol groups of biomolecules via maleimide conjugation. The significant advantage of our approach is that our viscosity probes can uniquely be positioned at the surfaces of either biomolecules or interfaces, and can subsequently report on local viscosity through either fluorescence emission or time-resolved fluorescence lifetime measurements.  Our probes are ideal for monitoring environmental changes, such as protein folding, small molecule binding etc.  All our reactive tag Viscosity Probes (Maleimide) are sold in 5 mg quantities. Larger quantities can be available on request to This email address is being protected from spambots. You need JavaScript enabled to view it.

Viscous Blue 2 Maleimide

Similar to the free fluorophore Viscous Blue 2™, Viscous Blue 2 Maleimide produces protein (and other) conjugates that can be excited from 300-420 nm, optimally at 360nm. Figure 1A, shows the absorption spectrum of Viscous Blue 2 Maleimide in Ethanol, Glycerol and Water. Viscous Blue 2 Maleimide is water / buffer soluble.

Viscous Blue 2 Maleimide

Figure 1. (A) Absorbance spectra recorded for Viscous Blue 2 Maleimide™ dissolved in ethanol (blue solid line), glycerol (red dash dotted line), and in water (black dotted line). (B) Excitation-Emission intensity Map (EEM) recorded for Viscous Blue 2 Maleimide™ dissolved in glycerol at + 20 °C (68 °F).

 

Figure 1B shows the excitation and emission intensity map. In neat solvents and unconjugated, the emission intensity maximum of Viscous Blue 2 Maleimide is centered around 450 nm, approximately 20 nm red shifted as compared to Viscous Blue 2™.

Viscous Blue 2 Maleimide is a viscosity sensitive probe, whose emission intensity increases with increasing viscosity. Figure 2 shows the emission intensity of Viscous Blue 2 Maleimide as a function of glycerol viscosity.

Viscous Blue 2 Maleimide

Figure 2. (A) Emission spectra recorded for Viscous Blue 2 Maleimide™ dissolved in neat glycerol at different temperatures, from 0 °C to + 80 °C. The excitation wavelength was 360 nm. (B) Integrated band intensity plotted as function of glycerol viscosity. .

 

 

Viscous Blue 420 Maleimide

Similar to Viscous Blue 420™, Viscous Blue 420 Maleimide produces protein (and other) conjugates that can be excited from 280-380 nm, optimally around 320 nm. Figure 3 A shows the absorbance spectrum recorded for Viscous Blue 420 Maleimide™ in Water, Ethanol and Glycerol. Viscous Blue 420 Maleimide™ is water / buffer soluble.

conjFigure 3

Figure 3. (A) Absorbance spectra recorded for Viscous Blue 420 Maleimide™ dissolved in ethanol (blue solid line), glycerol (red dash dotted line), and in water (black dotted line). (B) Excitation-Emission intensity Map (EEM) recorded for Viscous Blue 420 Maleimide™ dissolved in glycerol at + 20 °C (68 °F).

 

Figure 3B shows the excitation and emission intensity 3D map. In neat solvents and unconjugated, the emission intensity maximum of Viscous Blue 420 Maleimide™ is centered around 400 nm, very similar to the parent viscosity probe molecule, Viscous Blue 420™.

conjFigure 4

Figure 4. (A) Emission spectra recorded for Viscous Blue 420 Maleimide™ dissolved in neat glycerol at different temperatures, from +10 °C to +100 °C. The excitation wavelength was 330 nm. (B) Integrated band intensity plotted as function of glycerol viscosity. .

 

Viscous Blue 420 Maleimide™ is a water soluble viscosity sensitive probe, whose emission intensity increases with increasing viscosity. Figure 4 shows the emission intensity of Viscous Blue 420 Maleimide™ as a function of glycerol viscosity, where the glycerol viscosity has been changed with temperature.

Our Viscous Blue 420 Maleimide™ reactive tag viscosity probe belongs to the class of molecular rotors based on Twisted Intramolecular Charge Transfer (TICT) states. The accessibility of the dark non-emissive TICT state is typically responsible for the non-radiative decay pathways in the rotor, which can be altered via viscosity, giving rise to the viscosity dependence observed. Viscous Blue 420 Maleimide™ is ideal for conjugation to proteins and other biomolecules using well-known Maleimide reaction chemistries, providing local / surface viscosity measurements.

Labeling of Proteins / Biomolecules with Reactive Tag Viscosity Probes

Ursa Biosciences Maleimide reactive tag viscosity probes can be readily conjugated to a range of proteins, peptides and biomolecules. We offer a simple protocol below. However, the efficiency of labeling may ultimately depend on the properties of the proteins, biomolecules and peptides to be labeled, our protocol being a starting point for researchers less familiar with linking thiols to maleimides.

Ursa BioScience maleimide reactive tag viscosity probes are electrophilic compounds which show high selectivity towards thiols. Thiols in biomolecules are highly prone to oxidative dimerization, forming disulfide bonds. Disulfides however do not react with maleimides, therefore it is necessary to reduce disulfides prior to reaction with our reactive tag viscosity probes, in the absence of oxygen. For researchers conjugating to synthetic DNA which contains thiol groups, then no reduction may be needed.

The solubility of the starting materials is an important consideration in the conjugation protocol. Our malemide probes are soluble in water, although researchers may want to consider using deoxygenated DMF (fresh) or DMSO.

Step 1: In a plastic vial or tube, dissolve 1-10 mg/ml of the biomolecule, containing the thiol group to be labeled, in either degassed PBS or TRIS buffers at a pH between 7 and 7.5.

Step 2: Add an excess of TCEP (tris-carboxyethylphosphine) to the biomolecule solution to reduce the disulfide bonds. Typically a 75-125x molar excess of the TCEP is used. After addition of the TCEP, the tube should be flushed with either nitrogen or argon and sealed for 30 mins.

Step 3: The 5 mg of the reactive tag viscosity probe should be dissolved in 100 µL of either water, DMSO or DMF.

Step 4: The reactive tag viscosity probe solution should then be added to the reduced biomolecule solutions (now thiol groups), where the dye is approximately 20x fold in excess. The vial should be shaken vigorously and purged with either nitrogen or argon, sealed and left to stand for 12 hrs at 4 C.

Step 5: The mixture can then be purified by either gel filtration or HPLC to recover the viscosity probe labelled biomolecule.

Further notes:

1.    Degassing: Degassing to produce deoxygenated solutions can be achieved by bubbling either nitrogen or argon through the solution for 20 mins. The solution should then be sealed until use.

2.    Degassing: Degassing can also be achieved by applying a vacuum to the solution to be degassed, for several minutes.

3.    Purification: For water soluble maleimides and mixtures, dialysis can be used for purification.

4.    The molecular weight of Viscous Blue 420 Maleimide™ is: 305 g/mol, ε= 108, 630 mol-1 L cm-1 (ACN).

5.    The molecular weight of Viscous Blue 2 Maleimide™ is 290 g/mol.

 

Viscous Blue 2 Maleimide reaction