23


Alternatively, fl uorescent molecular ‘tags’ have been designed that can specifi cally bind molecules of interest, such as proteins or other targets, and the known fl uorescence profi le of these tags, or how those change when molecules interact can provide a lot of information. Applications of this technique include looking at cell viability, apoptosis, enzyme kinetics and oxidative stress, for example.


Choosing the right technique for biological applications


Applications of UV-Vis for biology


In many cases biological applications require working with nucleic acids or proteins and samples are in low volumes, are diffi cult to obtain or expensive. UV-Vis spectroscopy is a robust, informative technique that does not require a lot of sample and equally has a strong advantage of not requiring much sample preparation.


DNA and RNA Analysis: UV-Vis spectroscopy is commonly employed to verify the concentration and purity of DNA and RNA samples. This is particularly important for downstream applications such as sequencing. By assessing the absorbance patterns of these nucleic acids, researchers can ensure the quality and suitability of their samples for further analysis.


Growth of a bacterial culture for protein expression.


Applications of fl uorescence spectroscopy for biology


Fluorescence spectroscopy fi nds widespread applications in biological research.


While taking measurements, the cuvette should be placed in the cuvette holder such that the transparent sides are aligned with the beam path. If only a small amount of sample is available, consider a smaller volume cuvette to ensure that the sample is in the beam path (see Table 1).


Temperature (°C) Tm


Figure 1. The plot represents a melting curve of a DNA sample.


Figure 1: A wavelength scan of a DNA sample showing an absorbance level of around 0.5 Abs and 260 nm.


Protein Analysis: In biochemical and biophysical studies, UV-Vis spectroscopy measures the absorbance of proteins at approximately 280 nm due to the absorbance from tryptophan and tyrosine residues. For a pure protein solution, for example assessment of a protein drug, the presence of absorbance at 350 nm can indicate aggregation due to instability.


Bacterial Culture Studies: UV-Vis spectroscopy aids in monitoring bacterial growth and culture ‘optical density’. By measuring the absorbance of bacterial suspensions at approximately 600 nm, researchers can monitor cell proliferation and study the effects of various conditions on bacterial populations.


This document describes how to optimize experimental conditions for thermal melt measurements to achieve high-quality data with confidence. Here are our top tips:


Use the right cuvette


Enzymatic Reactions: Researchers use UV-Vis spectroscopy to monitor enzymatic reactions. Changes in absorbance during enzymatic processes provide insights into reaction kinetics, substrate utilisation, and enzyme inhibitor activity.


The choice of cuvette will depend on the volume of sample to be analyzed, the z-height of the UV-Vis spectrophotometer1 and the wavelengths used. The most widely used cuvette in UV-Vis spectroscopy has a pathlength of 10 mm and volume of 3.5 mL.


Thermal melt measurements are typically done at UV wavelengths and therefore the cuvette needs to transmit UV light. Quartz cuvettes are recommended for this reason.


Figure 2. The highly focused beam of the Cary 3500. Figure 2: An example of Time Resolved FRET.


If measuring more than one sample at a time, it is recommended to use the same type of cuvette in all the cuvette positions. Heat transfer from the Peltier heating elements to the sample depends on the physical characteristics of the cuvette, so using different cuvette types can introduce an unwanted variable.


Binding Studies: Fluorescence allows us to explore ligand- receptor interactions. When a ligand binds to a receptor (e.g., drug-receptor binding), the fl uorophore’s environment changes, affecting its emission properties. Binding constants and dissociation rates can be determined precisely.


The melting profile of other molecules/compounds can also be analyzed using UV-Vis spectroscopy. Some examples include polymer cloud-point determination, drug-protein interactions, and protein thermal denaturation.


The Cary 3500 system is ideal for measuring small volume nucleic acid samples as the instrument has a highly focused beam of less than 1.5 mm width (see Figure 2). The instrument’s factory-aligned optics require no adjustment before taking measurements, even when measuring multiple cuvettes at the same time.


Molecular Probes: Fluorescent dyes and probes are essential tools for studying biomolecules. These probes selectively bind to specifi c targets (e.g., proteins, nucleic acids) and emit fl uorescence upon binding. Researchers use them for localisation studies, tracking cellular processes, and detecting molecular interactions.


Protein Folding and Dynamics: Fluorescence sheds light on protein folding pathways and dynamics. By attaching fl uorophores to specifi c amino acids, scientists monitor conformational changes in how these molecules interact. Förster resonance energy transfer (FRET) between two fl uorophores provides distance information, revealing protein fl exibility and stability.


Absorbance (A)


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88  |  Page 89  |  Page 90  |  Page 91  |  Page 92  |  Page 93  |  Page 94  |  Page 95  |  Page 96  |  Page 97  |  Page 98  |  Page 99  |  Page 100  |  Page 101  |  Page 102  |  Page 103  |  Page 104  |  Page 105  |  Page 106  |  Page 107  |  Page 108  |  Page 109  |  Page 110  |  Page 111  |  Page 112  |  Page 113  |  Page 114  |  Page 115  |  Page 116  |  Page 117  |  Page 118  |  Page 119  |  Page 120  |  Page 121  |  Page 122  |  Page 123  |  Page 124  |  Page 125  |  Page 126  |  Page 127  |  Page 128  |  Page 129  |  Page 130  |  Page 131  |  Page 132  |  Page 133  |  Page 134  |  Page 135  |  Page 136  |  Page 137  |  Page 138  |  Page 139  |  Page 140  |  Page 141  |  Page 142  |  Page 143  |  Page 144  |  Page 145  |  Page 146  |  Page 147  |  Page 148