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Cysteine (Cys) and Homocysteine (HCys) Probes

Ursa BioScience offers a range of patent protected water soluble, high quantum yield and highly photostable probes for both the rapid detection and quantitation of Cysteine (Cys) and Homocysteine (HCys). Our probes are readily able to detect both Cys and HCys in i) both an absorption / excitation spectrum and ii) a Fluorescence manner.

Our probes are easy to use, provide highly reproducible results and are supplied with detailed data sheets.

Cysteine Green™

Cysteine Green™ is highly selective probe for both the detection and quantitation of both Cysteine (Cys) and Homocysteine (HCys). Cysteine Green™ is readily soluble in aqueous buffers such as PBS (Phosphate Buffered Saline) and in MeOH or EtOH, with an extinction coefficient of ≈ 19753 M-1 cm-1 in EtOH and 40498 M-1 cm-1 in PBS. The Mw of Cysteine Green™ is 385.3 g/mol. Cysteine Green has an absorption and emission spectrum very similar to that of Fluorescein, enabling standard fluorescein filters and microscope dichroics to be used with Cysteine Green™. When bound to Cysteine in PBS buffer, Cysteine Green™ has a quantum yield of ≈ 0.298.

Cysteine Green™ shows a time dependent interaction with L-Cysteine, with both the absorption and excitation spectra band centered at ≈ 500 nm, increasing over time and increasing with higher concentrations of Cysteine (and Homocysteine).

Figure 1: (A) Absorbance spectra recorded as a function of time, 0 to 80 min, for a 1.0 μM sample of Cysteine Green™ mixed with 8.0 mM L-Cysteine in phosphate buffered saline (PBS). The arrows indicate spectral trends with increasing time. (B) Peak absorbance observed at ~500 nm plotted as a function of time for different concentrations of L-Cysteine. The trace recorded for 8.0 mM L-Cysteine, i.e. the blue curve, corresponds to data presented in panel A. Note that the concentration of Cysteine Green™ was kept fixed at 1.0 μM for all samples.

 

Similar to the absorption spectrum the excitation spectrum also shows a notable increase in intensity as a function of i) the concentration of either Cysteine or Homocysteine and ii) as a function of time. The response is quite pronounced, with the dynamic response range being greater using the excitation spectrum as compared to the absorbance spectrum. The excitation spectrum is recorded by holding the emission wavelength constant (here at 525 nm) and scanning the excitation wavelengths, (here from 400 – 510 nm). For cysteine and homocysteine sensing, the user can simply excite at 490 nm and monitor the emission at 525 nm, a very simple 2 point measurement, data similar to figure 2B being produced.

Figure 2

Figure 2: (A) Excitation spectra recorded as a function of time, 0 to 70 min, for a 1.0 μM sample of Cysteine Green™ mixed with 10.0 mM L-Cysteine in phosphate buffered saline (PBS). The arrow indicates the spectral trend with increasing time. The emission monochromator was set to 525 nm. (B) Emission intensity, here the area indicated grey between 490 and 500 nm in panel A, plotted as a function of time for different concentrations of L-Cysteine. The trace recorded for 10.0 mM L-Cysteine, i.e. the blue curve, corresponds to data presented in panel A. Note that the concentration of Cysteine Green™ was kept fixed at 1.0 μM for all samples.

 

The concentration of either Cysteine or Homocysteine can readily be determined using simple calibration plots, as shown in Figure 3, obtained from the excitation spectra.  Here we have simply plotted concentration Vs time, data from Figure 2B. By simply choosing a time point suitable for your experiments, you can also generate calibration plots similar to these.

Figure 3: Intestines observed as a function of concentration L-Cysteine at three different times, as presented in Figure 2 B.

 

Quantitation of either Cysteine or homocysteine levels in your samples, can additionally be achieved by measuring the fluorescence enhancement at a particular time point, such as 5, 10 or 20 mins after the addition of Cysteine Green™ to your sample, and comparing to a calibration curve. Our data sheets describe how to make your own calibration curves for both Cys and HCys in your sample of choice.

Figure 4: Comparison of time-dependent intensities observed at 525 nm, excitation at 495 nm, for Cysteine Green™ when mixed with L-Cysteine or L-Homocysteine in PBS at 5 mM concentration. The concentration of dye was 1.0 μM.

 

Cysteine Green™ is a specific probe for the detection of either Cysteine or Homocysteine. After complete reaction, i.e. after no further change in the fluorescence emission spectrum, Cysteine Green™ shows a 1.43 fold selectivity for Cys over that of HCys, the quantum yields of the reaction products being 0.289 and 0.202 respectively. The Cys reaction with Cysteine Green™ is also much faster than the reaction of HCys with Cysteine Green™ in the early part of the reaction, allowing the user to readily select for Cys over HCys.

The response of Cysteine Green™ to both Cys and HCys is linear over the range 100 µM ⟶ 1 mM, with the selectivity towards Cys greater than 2-fold for concentrations as low as 100 µM. We estimate that the lower limit of detection for Cys is ≈ 3-fold lower than HCys, (39 µM Vs 114 µM after 20 mins, 1 µM Cysteine Green™). With regard to other interferences, the observed trend for fluorescence enhancement is Cys (40.98 fold) > HCys (28.73 fold) > mercaptoethanol (6.04-fold) > mercaptopropionate (1.66-fold), after complete reaction with the respective thiol. Cysteine Green™ also shows fluorescence enhancement for Glu-Cys (a C-terminal cysteine dipeptide) and laminin 11 (a synthetic nonapeptide containing an N-terminal cysteine residue, also known as peptide 11, often used in the development of anti-metastatic drugs).

Figure 5: (A) Emission intensities observed as function of time for Cysteine Green™ mixed with various concentrations L-Cys in PBS, recorded in an excitation spectra manner. The concentration of dye was kept fixed at 1.0 µM. The sample was excited at 495 nm and the emission observed at 525 nm. (B) Emission signal observed at 22 minutes plotted as function of concentration L-Cys.

 

In addition to Absorbance measurements or using the Excitation spectrum for sensing, Cysteine Green™ can also be used in a traditional fluorescence manner, and is highly effective for determining Cys and HCys levels. Cysteine Green can be excited from 400 – 500 nm, with an emission centered around 520 nm.

Figure 6: (A) Emission spectra recorded as a function of time, 0 to 40 min, for Cysteine Green™ at a 1.0 μM concentration mixed with 1.0 mM L-Cys in PBS. The sample was excited at 460 nm. (B) Emission signal, here the grey area in panel A, plotted as a function of time for different concentrations L-Cys.

 

Cysteine Green™ shows a bioexponential fluorescence decay in PBS (Phosphate Buffered Saline) buffer, τ1 = 2.22 ns (24.5%), τ2 = 3.95 ns (75.5 %) with an amplitude weighted lifetime of ≈ 3.31 ns, which decreases to ≈ 3.15 ns in the presence of 500 µM L-Cys after 60 minutes incubation (plateau value), λex = 467 nm, λem = 510 nm.

Figure 7: (A) Time-resolved decay recorded for Cysteine Green™ at 1.0 µM concentration in PBS. The excitation wavelength was 467 nm and the emission observed through a monochromator centered at 510 nm. (B) Same sample as presented in panel A, but after the addition of 500 µM L-Cys. The sample was equilibrated for 60 minutes before data were recorded.

 

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Cysteine Green™ in Pre-Loaded Cuvettes

We offer Cysteine Green™ already conveniently loaded into a box of 100 plastic cuvettes (transmission down to 285 nm). These cuvettes allow for Cys or HCys detection / determination in either an absorption or fluorescence based format. With these convenient pre-loaded cuvettes, there is no reconstitution procedure necessary, simply add 3 ml (3cm3) of your sample to each cuvette and measure, it’s that simple.

Go to the online store for a price for this kit.

 

Dual Cys™

Dual Cys™ is a remarkable fluorescent probe that is simultaneously sensitive to both Cysteine (Cys) and Homocysteine (HCys). This fluorescent probe has 2 unique fluorescence bands, one at 380 nm, the other at 487 nm, which both show a turn on fluorescence signal (i.e. it gets brighter) when either Cys or HCys is present. What is remarkable is that the fluorophore contains functionalities that respond to both Cys and HCys, from the same molecule. This dual functionality for both Cys and HCys, is not offered by any other commercial fluorophore product today and is only available from Ursa BioScience™. The ability to measure both Cys and HCys by the same molecule in the same spatial location, is ideal and unique for studying cellular processes.

Dual Cys™ is readily soluble in aqueous buffers such as PBS (Phosphate Buffered Saline) and in MeOH or EtOH, with an extinction coefficient of ≈ 24 000 M-1 cm-1.

Figure 8: (A) Absorption spectra recorded for Dual Cys™ dissolved in ethanol at different concentrations. (B) Peak absorption observed at 299 nm plotted as a function of the concentration of the dye. The molar extinction coefficient is calculated from the slope to ≈ (25 000 ± 1 500) M-1 cm-1. The error accounts for weighing and dilution inaccuracies. Note that the error specified in the graph is for the regression only.

 

Dual Cys™ is highly selective probe for both the detection and quantitation of Cysteine (Cys) and Homocysteine (HCys). It can readily be excited from 250 – 340 nm. The probe is highly photostable.

Figure 9: (A) Contour emission graph recorded for a sample containg 20 μM Dual Cys™  dissolved in EtOH/phosphate buffer (20 mM, pH 7.4, 2:8 v/v) (B) Same sample as presented in panel A, but with 1 equivalents of L-cys added.

 

Dual Cys™ not only detects both Cysteine (Cys) and Homocysteine (HCys) at the same time (simultaneously), but it can also easily be used to quantitate both in the same sample. The fluorescence band at 380 nm readily increases in intensity as a function of the increased concentration of Homocysteine in the sample. Likewise, the Cysteine band at 487 nm, also increases in intensity (gets significantly brighter) as a function of the increasing concentrations of Cysteine in the sample. This dual function behavior allows both Cys and HCys to be analytically quantitated in samples. The calibration plots in panels B and D of figure 10, readily allow the Cys and HCys to be determined, respectively. Concentrations < 1 µM can be determined using Dual Cys™.

Figure 10: Emission spectra recorded for samples containing 20 μM Dual Cys™ mixed with different concentrations of either L-Cysteine (A,B) or Homocysteine (C,D). The spectra were recorded 40 minutes after the initial mixing. Panels B and C show calibration curves constructed by plotting the peak maxima, 487 nm and 380 nm for L-Cysteine and Homocysteine, respectively, versus concentration. The solvent used is EtOH/phosphate buffer (20 mM, pH 7.4, 2:8 v/v).

 

Figure 11: (A) Time-resolved intensity decay recorded in the magic angle format for a sample containing 20 μM Dual Cys™ dissolved in EtOH/phosphate buffer (20 mM, pH 7.4, 2:8 v/v) with 1 equivalents of L-Cys added. The excitation wavelength was 311 nm and the emission collected at 480 nm using a monochromator. The technique used to record data is the Time-Correlated Single Photon Counting (TCSPC) method. The data fits well to a 2-exponential decay function as supported by inspecting the weighted reiduals and the reduced Chi Squared value. The fitted decay constants and their contribution to the intensity are 0.63 ns (89 %) and 1.25 ns (11 %). (B) Contour emission graph recorded for the sample used for the TCSPC experiment with the data collection region indicated.

 

Dual Cys™ can be purchased in the On Line store.

 

Dual Cys™ in Pre-Loaded Cuvettes

We offer Dual Cys™ already conveniently loaded into a box of 100 plastic cuvettes (transmission down to 285 nm). These cuvettes allow for Cys or HCys detection / determination in either an absorption or fluorescence based format. With these convenient pre-loaded cuvettes, there is no reconstitution procedure necessary, simply add 3 ml (3cm3) of your sample to each cuvette and measure, it’s that simple.

Go to the online store for a price for this kit.

 

 

 

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Homocysteine

 

 

180px-Homocysteine racemic

 

 

Cysteine green contourv2

Contour emission graphs recorded for Cysteine Green™ before (A) and after (B) L-Cysteine is added. The concentration L-Cysteine is 10 mM and the solvent PBS. After L-Cysteine was added the sample was left to equilibrate for 60 minutes.

 

green

The Cysteine Green™ cuvette kit contains 100 plastic cuvettes already preloaded with Cysteine Green™.  Simply add 3 ml of your sample to each cuvette.

 

Ursa standard prodcut vialCysteine Green™ can be supplied dry in a prepacked vial for easy reconstitution. Visit our Online Store for current pricing. Watch our online video for our reconstitution procedure.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mg 510

Cuvettes containing ~20 μM Dual Cys™ dissolved in EtOH/phosphate buffer (pH 7.4, 2:8 v/v). The cuvettes are placed on an UV light box and illuminated from below at 365 nm. The cuvette on the right contains an additional 20 μM L-Cysteine. i.e. 1 equivalents. Corresponding contour emission graphs are shown in Figure 9.

 

Ursa standard prodcut vial

Dual Cys™ is supplied dry in a prepacked vial for easy reconstitution. Visit our Online Store for current pricing. A short video shows our reconstitution procedure.

 

 

Mg 510

Cuvettes containing ~20 μM Dual Cys™ dissolved in EtOH/phosphate buffer (pH 7.4, 2:8 v/v). The cuvettes are placed on an UV light box and illuminated from below at 365 nm. The cuvette on the right contains an additional 20 μM Homocysteine, i.e. 1 equivalents.

 

Mg 510

The Dual Cys™ cuvette kit contains 100 plastic cuvettes already preloaded with Dual Cys™.  Simply add 3 ml of your sample to each cuvette and measure. See our online store for a price for this kit.