Основні поняття
In-gel fluorescence (IGF) detection is a rapid, cost-effective, and quantitative alternative to immunoblotting for visualizing endogenously expressed fluorescent proteins in cell extracts.
Анотація
The study demonstrates that various green and red fluorescent proteins (FPs) can maintain their native, fluorescent form during the entire process of protein sample extraction, incubation with sample buffer, and migration on SDS-PAGE with only minor adaptations of traditional protocols. This allows the direct detection and quantification of in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs using standard fluorescence-imaging devices.
The authors systematically compare the suitability of different FPs, including yeGFP, EGFP, sfGFP, mNeonGreen, mCherry, mRuby2, and TagRFP-T, for IGF detection. They evaluate key parameters such as fluorescence intensity, heat-stability, and overall compatibility with standard SDS-PAGE protocols and imaging devices. The results show that EGFP, sfGFP, and mCherry are particularly well-suited for IGF detection, with mCherry exhibiting exceptional heat-stability.
IGF detection eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent to immunoblotting such as transfer onto a membrane and antibody incubations. The authors demonstrate that IGF provides clearer data with less background interference, sensitivity comparable or better to antibody-based detection, better quantification, and a broader dynamic range. After fluorescence imaging, the gels can still be used for other applications such as total protein staining or immunoblotting if needed.
The study also explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts from various organisms, including yeast, Drosophila, MDCK cells, and C. elegans. The authors provide insights into sample preparation, imaging conditions, sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.
Статистика
"Bmh1-yeGFP and Hxk1-yeGFP migrated faster than when samples were heated at 95°C or precipitated with TCA, as determined after transfer and immunoblotting with anti-Bmh1 and anti-Hxk1/2 antibodies."
"Bmh1-yeGFP fluorescence signal could be detected on the nitrocellulose membrane even after transfer and incubation with antibodies, allowing the sequential observation of yeGFP fluorescence and antibody-based labeling during the same acquisition."
"The temperature at which 50% of the signal is still present was 67°C for sfGFP and 59°C for yeGFP."
"The temperature at which 50% of the signal is still present was 79°C for mCherry."
Цитати
"IGF detection provides clearer data with less background interference, a sensitivity comparable or better to antibody-based detection, a better quantification and a broader dynamic range."
"Increasing the sample pH to pH 8.0 protects GFP denaturation in diluted samples."
"Overall, IGF detection is also a cost-effective method. This technique eliminates the need for specific antibodies, which may even not be available in the case of less conventional FPs, as well as nitrocellulose membrane or ECL reagents."