UV-visible microspectroscopy is an important tool in the analysis of microscopic protein crystals, which presently are used in a wide variety of applications ranging from drug design research and structural biology through to bio-separation of drugs such as insulin and controlled drug delivery systems.
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A vapor diffusion process, such as the hanging or sitting drop method, is used to grow most protein crystals. However, salt crystals are also found frequently alongside these crystals. Normal imaging methods can make it difficult to distinguish these two crystals, but protein readily absorbs light at 280 nm and fluoresces in the near UV.
While protein can be differentiated from the salt crystals by imaging the intrinsic protein fluorescence, the process is quite slow and provides very little information, apart from being susceptible to contamination. For example, even the well plates in which the crystals are grown may exhibit fluoresce. On the other hand, ultraviolet microspectroscopy is fast, reliable and provides much more information about the crystal, such as protein concentration in the crystal.
CRAIC Technologies™ Inc. has developed UV microspectrophotometers, such as the 20/20 PV™ that can quickly differentiate protein from salt crystals through absorbance and fluorescence microspectroscopy.
Apart from this, UV microspectrophotometers can also proceed to qualify the crystal after it has been identified and located. The test procedure is simple, in which a spectrum of a microscopic crystal is acquired. At 280 nm, protein crystals absorb the light strongly. If the crystal is a salt, it does not absorb the 280 nm light. Moreover, the use of spectra makes it possible to determine the protein concentration in the crystal as well as the potential contamination of the crystal.
An example of the UV-visible absorbance microspectra of a single microscopic protein crystal is shown below. All measurements were carried out using a UV-visible-NIR range microspectrophotometer from CRAIC Technologies™. A UV transparent hanging drop well plate was used to grow the crystal and the spectra of the crystal were taken while it was still in the plate. In order to show the clear difference between protein and salt crystals at 280 nm, images of the protein crystals were also captured.
Figure 1. Absorbance spectrum of single protein crystal from which concentration of protein in the crystal could be determined. The single peak is indicative of a pure protein crystal.
Figure 2. UV microscope image of protein crystal. Salt crystals are clear.
This information has been sourced, reviewed and adapted from materials provided by CRAIC Technologies.
For more information on this source, please visit CRAIC Technologies.