Abstract
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized technique for macromolecule and protein analysis. While multiple methods exist to visualize the separated protein bands on gels, one of most popular methods of staining the proteins is with Coomassie dye. A more recent approach is to use Bio-Rad stain-free technology for visualizing protein bands with UV light and achieve similar or greater sensitivity than that of Coomassie dye. Here, we developed a method to further amplify the sensitivity of stain-free gels using carboxyfluorescein succinimidyl ester (CFSE) staining. We compared our novel method using foetal bovine serum samples with Coomassie dye, standard stain-free gels, and silver staining. Our results show that while silver staining remains a gold-standard method in terms of sensitivity; CFSE staining of samples prior to use with stain-free gels results in a 10–100-fold increase in sensitivity over Coomassie staining and the standard stain-free method. Our method offers a sensitivity similar to that of silver staining which is compatible with downstream mass spectrometry, and therefore more advantageous for further retrieval and analysis of macromolecules in bands.
Highlights
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized technique for analyzing macromolecules such as proteins and nucleic acids [1,2,3]
They were trapped on a peptides were solubilized in 2% acetonitrile, 0.5% acetic acid, 97.5% water for mass spectrometry trapping column and separated on a μm PepMap reverse phase column analysis; percentages refer to volume per volume
Unstained fetalbovine bovineserum serum (FBS) and carboxyfluorescein succinimidyl ester (CFSE)-stained FBS samples ranging five orders of magnitude in protein quantity were run on gels with silver staining, Coomassie staining, and stain-free methodologies, Figure 3
Summary
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized technique for analyzing macromolecules such as proteins and nucleic acids [1,2,3]. The staining protocol is time-consuming and results are affected by a number of factors such as reagent quality, incubation times, and gel thickness [3] Another drawback of many of these stains is that many are not compatible with downstream analysis methods such as Western blotting and mass spectrometry [6]. While stain-free technology is designed to work without additional protein staining, it is possible to prelabel protein samples with various fluorophores to increase their signal intensity One such compound of interest is carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFSE exhibits a relative excitation intensity of around 50% at UV wavelengths (emission peak at 517 nm) [9] This property makes it a promising compound for enhancing protein detection in stain-free gels, which are activated by UV light. We demonstrate a method of combining CFSE labeling with stain-free gel technology to enhance the sensitivity of detection for protein samples
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