Automatically scan fluorescent protein molecules to determine their optimal wavelengths with the SpectraMax Paradigm microplate assay platform and Tune technology

Automatically scan fluorescent protein molecules to determine their optimal wavelengths with the SpectraMax Paradigm microplate assay platform and Tune technology

Based on the SpectraMax® Paradigm® multi-function microplate inspection platform, Molecular Devices has introduced a Tune cartridge that uses a filter as its monochromator but can adjust its detection wavelength at will. The technology combines the high sensitivity of the filter with the high flexibility of wavelength scanning. The Tune test cartridge overcomes the shortcomings of traditional grating-type monochromators, allowing researchers to optimize the optimum wavelengths required for various tests over a wider range of wavelengths, and the detection sensitivity is more than 10 times higher than that of the grating system. . The Tune cartridge emits light with a wavelength range of 360nm to 790nm, an emission wavelength range of 400nm to 850nm, and can be stepped in 1nm. It is also a multi-function detection cartridge that has time in addition to the fluorescence intensity (FI) detection mode. Resolution fluorescence (TRF) and chemiluminescence (Lum) detection modes.

SpectraMax Paradigm inspection platform incorporates new Tune detection technology

The patented Spectral Optimization Wizard simplifies the workflow by automatically calculating the ratio of signal to background for different fluorescent dye molecules after scanning, to find the optimal excitation and emission wavelength for this fluorescent dye molecule. The software will automatically generate a scanning heat map, and give the calculated signal to background ratio according to different scanning wavelengths, which is convenient for the user to select the optimal wavelength to complete the experiment. When the wavelength is automatically optimized using EGFP fluorescent dye molecules, the detection sensitivity is More than 80 times better than using traditional fixed wavelength methods .

Excitation and emission spectroscopy

Individually scanning excitation and emission wavelengths for the two mutants of the green fluorescent protein, respectively, the recombinant wild-type GFP (Clontech) and enhanced GFP (EGFP, BioVision) are TE buffer were diluted to 10μg / ml using, PH adjusted to 8 . To measure the spectroscopy properties of the GFP sample ( 50 ul ), it was added to a black 384- well plate, and the well containing only buffer in the black microplate was used as a reference well.

When performing separate excitation and emission spectral scans, set the Integration Time to 140ms . When the optimum excitation wavelength is determined for scanning, the emission wavelength is first set to 530 nm , the excitation wavelength is from 360 nm to 500 nm, and every 2 nm steps. When the optimum emission wavelength is determined for scanning, the excitation wavelength is first set to 440 nm , the emission wavelength is from 470 nm to 600 nm, and every 2 nm step. Figure 1 shows that when the emission peak of EGFP is near 506 nm ( GFP ), i.e. , 512 nm (EGFP ; Figure 1 ) , its excitation peak is red-shifted from 394 nm to 482 nm .

GFP and EGFP use Tune technology (Figure 1)

Spectra of GFP and EGFP were obtained in the SoftMax Pro6 software spectral scan mode using the SpectraMax Paradigm microplate assay platform and Tune technology . We clearly see from the spectrum that the maximum value of their excitation peaks shifts, the blue line represents the GFP spectrum, the green line represents the EGFP spectrum, the dashed line represents the excitation spectrum, and the solid line represents the emission spectrum.

Spectrum Optimization Wizard

Using the Spectral Optimization Wizard feature in SoftMax Pro6 software to determine the optimal wavelength for GFP and EGFP , the user can select the range of excitation and emission wavelengths, as well as the wavelength step. The Spectral Optimization Wizard scans the excitation and emission wavelengths simultaneously and calculates the ratio of the signal to the background for each pair of excitation / emission waves. The formula for the calculation is (signal - background) / background. The results show a pair of excitation / emission wavelengths in the form of a heat map. The position of the center of the cross icon shows the highest signal to background ratio. Figure 2 shows the GFP scan results.

Optimize excitation and emission wavelengths (Figure 2)

The results of the spectral optimization wizard can be displayed in the form of a heat map, giving the optimal excitation and emission wavelengths of the GFP fluorescent dye molecules, showing the optimal excitation wavelength of 405 nm , emission wavelength 510 nm , and optimal excitation wavelength of EGFP after GFP optimization. 465nm , emission wavelength 515nm ( not shown )

From the data in Table 1 , it can be found that the sensitivity of various mutants of GFP is improved by using the wavelength optimization guide function . The GFP excitation peak is 112 nm away from the emission peak . The sensitivity is improved by the wavelength optimization. The excitation and emission peaks are moderately changed. However, the EGFP excitation peak is only 30 nm away from the emission peak . The optimal wavelength can be calculated by the spectral optimization wizard function. The detection sensitivity can be increased by 80 times, and the excitation and emission peaks are optimized to be separated by 50 nm , which reduces the excitation light interference.

Table 1: Optimize the wavelength or fixed excitation and emission peaks through the Tune cassette , respectively , and compare the two methods for the detection of GFP and EGFP dye molecules (LLDs)

in conclusion

When the user is faced with a new unknown fluorescent dye molecule for wavelength optimization, the Tune cartridge's spectral optimization wizard function not only automatically optimizes the optimal excitation and emission wavelength of the dye molecule and can reduce the time by 50% . The excitation wavelength range is from 360nm to 790nm and the emission wavelength range is from 400nm to 850nm . The SpectraMax Paradigm microplate inspection platform and Tune cartridge can detect most commonly used fluorescent dye molecules and have higher detection sensitivity than traditional grating systems. More than double. As shown in the above test, when the Stokes shift of the fluorescent dye molecules ( such as EGFP) used is narrow, the sensitivity of the test detection can be greatly improved (up to 80 times) by the Tune cassette wavelength optimization function . In addition to the fluorescence intensity detection mode, the Tune cartridge also provides time-resolved fluorescence and chemiluminescence detection modes, increasing the variety of test detection methods.