Towards Informed Labelling & Purification Practices: How Transparency Impacts Workflows

For decades, molecular scientists have adhered to often cumbersome practices of purifying samples before conducting fluorescence-based assays, with a goal of eliminating unreacted fluorophores, ensuring the purity and reliability of experimental data.

A transformative shift is underway, as Labelling Quality Control reduces the frequency of required laborious purification.

Unreacted fluorophores

Conventional fluorescence-based assays, while invaluable, often grapple with a hidden challenge – the presence of unreacted fluorophores concealed within fluorescently labelled samples. This inconspicuous residue of free fluorophores can confound measurements of the intended labelled constructs, leading to skewed results and diminished sensitivity. In this article, we delve into the importance of labelling efficiency, explore methods for addressing the issue of unreacted fluorophores, and introduce a contemporary approach that revolutionizes how researchers deal with this fundamental, yet frequently overseen, concern.

Why is labelling efficiency a key QC parameter?

The presence of free fluorophore in the sample interferes with the measurement of the labelled construct, leading to inaccurate results and loss of sensitivity. To reveal the true picture, unreacted fluorophore must be either removed from the sample or accounted for during data analysis. ?

Traditionally, free fluorophores were removed via a clean-up step. However, this approach comes at the cost of time and sample waste. In some cases, it can even distort the native state of the labelled biomolecule, which is a major drawback of subsequent clean-up. For an accurate representation of the protein in its native state, measurements should ideally be conducted without disrupting its environment.

Modern technology offers an alternative paradigm. Instead of laboriously removing unreacted fluorophores, FIDA measurements embrace their presence. FIDA technology distinguishes between free and conjugated fluorophores in labelled samples, enabling precise adjustments during data analysis.

How are free and conjugated fluorophores in labelled samples distinguished?

FIDA is based on absolute measurement of size, leveraging the principle of molecular diffusion. By segregating measurements taken on free and conjugated fluorophores, data originating from free fluorophores is effectively isolated, allowing for analysis solely focused on the signals emitted by the labelled protein.

Species of different sizes are easily detected in the raw signal. The (automatic) peak area analysis in FIDA software identifies the fractions of free and conjugated fluorophore in a sample based on absolute molecular size. By separating the measurements made on free and conjugated fluorophore, data arising from free fluorophore can be removed and analysis is performed on only signal arising from the labelled protein.

Species of different sizes are easily detected in the raw signal. The (automatic) peak area analysis in FIDA software identifies the fractions of free and conjugated fluorophore in a sample based on absolute molecular size. By separating the measurements made on free and conjugated fluorophore, data arising from free fluorophore can be removed and analysis is performed on only signal arising from the labelled protein.

In what applications should labelling efficiency be measured?

Not all labelling strategies are created equal, and some will be better suited and more efficient in certain situations. Understanding the best option for your biomolecule is important to create the most productive assay. With FIDA technology, the labelling efficiency of your biomolecule of interest is easily and quickly revealed by providing an explicit ratio of free versus conjugated fluorophore with every measurement. If your biomolecule fails to reliably bind with the fluorophore, strategic optimization of the dye choice, labelling strategy, and sample condition can be performed to increase efficiency of labelling and thus the assay itself.

What kind of fluorophores can I use with a FIDA measurement?

FIDA technology is compatible with all types of labelling strategies: fusion constructs, covalent labeling, and non-covalent labeling. Additionally, the Fida 1 houses three excitation wavelengths to provide the most flexibility when choosing fluorophores: 480 nm for common fluorescent tags and dyes, 640 nm for increased sensitivity and reduced background fluorescence, and 275 nm for label-free samples. Fida 1 can switch excitation wavelengths quickly and easily in only 5 minutes, allowing the option to mix fluorophores and run labelled and label-free assays using the same instrument.

Fida 1 can switch excitation wavelengths quickly and easily in only 5 minutes, allowing the option to mix fluorophores and run labelled and label-free assays using the same instrument.

Is it acceptable to bypass the traditional purification step?

Purification has long been seen as a standard practice in molecular science, and many have accustomed to rigorous purification protocols. For those, it is natural to have doubts about leaving unreacted fluorophores in the samples. ?To address these concerns, it is crucial to recognize that FIDA technology operates on a fundamentally different principle. It is a first principle technology, capable to reach the level of precision of measurement of molecular size needed to distinguish between free and conjugated fluorophores. By utilizing absolute measurements and sophisticated data analysis techniques, FIDA technology can confidently quantify and account for free fluorophores, ensuring that your final results are based solely on the signals originating from the labelled protein. In essence, FIDA technology provides a means to embrace the presence of unreacted fluorophores while maintaining the integrity of your experimental system.

Learn more: https://www.fidabio.com/contact-us/discovery-call