Dark Reader - Reduce Photo-bleaching

Method
The photo-bleaching of SYPRO Orange-stained proteins by UV and DR light was measured. An SDS polyacrylamide gel was loaded with 3 aliquots of protein molecular weight standards (15 ng per band), subjected to electrophoresis and then stained with SYPRO Orange. Two of the protein lanes were cut out from the gel and exposed on either a 312 nm UV transilluminator (UV) or a DR transilluminator (DR) for 8 minutes. The protein lanes were then all photographed together on a DR transilluminator and the intensities of the fluorescent bands measured using IGOR 4.0 image analysis software.

Minimize Bleaching
The photo-bleaching of fluorophors upon exposure to light can become a significant problem, particularly when the experimental protocol is prolonged. This situation arises, for example, when proteins are being isolated from 2-D electrophoresis gels for downstream analysis. Clearly, if photobleaching can be minimized then the usable life of a gel can be extended accordingly, without the need to re-stain the gel. To determine the extent of photobleaching that occurs upon exposure of SYPRO Orange- stained proteins to DR and UV light, samples were variously exposed for 8 minutes on either a DR or a 312 nm UV transilluminator. The results below show that UV exposure causes a ~40% decrease in the fluorescence intensity of the protein bands. Interestingly, some proteins appeared to be more significantly affected than others and were almost undetectable after 8 minutes of UV exposure. The DR exposure, on the other hand, resulted in only a ~10% or less decrease in band intensity, indicating that the DR transilluminator is a more appropriate device for procedures that require extended exposure to exciting light.

	Method The photo-bleaching of SYPRO Orange-stained proteins by UV and DR light was measured. An SDS polyacrylamide gel was loaded with 3 aliquots of protein molecular weight standards (15 ng per band), subjected to electrophoresis and then stained with SYPRO Orange. Two of the protein lanes were cut out from the gel and exposed on either a 312 nm UV transilluminator (UV) or a DR transilluminator (DR) for 8 minutes. The protein lanes were then all photographed together on a DR transilluminator and the intensities of the fluorescent bands measured using IGOR 4.0 image analysis software.



	Clare Chemical Research