Maintaining Fluorescence of Endogenously Expressed Proteins During SDS-PAGE for Rapid and Cost-Effective Detection

Belangrijkste concepten

In-gel fluorescence (IGF) detection is a rapid, cost-effective, and quantitative alternative to immunoblotting for visualizing endogenously expressed fluorescent proteins in cell extracts.

Samenvatting

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.

Statistieken

"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."

Citaten

"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."

Belangrijkste Inzichten Gedestilleerd Uit

Direct observation of fluorescent proteins in gels: a rapid cost-efficient, and quantitative alternative to immunoblotting

by Sanial,M., M... om www.biorxiv.org 06-01-2024

https://www.biorxiv.org/content/10.1101/2024.05.31.594679v1

Diepere vragen

How could the IGF detection method be further optimized to minimize the bleed-through of signals when visualizing green- and red-tagged proteins in the same sample?

To minimize the bleed-through of signals when visualizing green- and red-tagged proteins in the same sample during IGF detection, several optimization strategies can be implemented:

Filter Selection: Utilize imaging systems with appropriate filters that have minimal overlap between the excitation and emission spectra of the green and red fluorescent proteins. This can help reduce the bleed-through of signals between the two channels.

Sequential Imaging: Consider sequential imaging of the gel for each fluorescent protein separately. By capturing images for green-tagged proteins first and then red-tagged proteins, the potential bleed-through can be minimized as each channel is imaged independently.

Image Processing: Employ image processing techniques to digitally separate the signals of green and red fluorescent proteins post-imaging. This can involve background subtraction, spectral unmixing algorithms, or software tools designed to separate overlapping signals.

Optimized Sample Preparation: Ensure uniform and consistent sample preparation techniques to minimize variations in fluorescence intensity that could contribute to bleed-through. Consistent protein loading and gel electrophoresis conditions can help reduce signal overlap.

Negative Controls: Include appropriate negative controls in the experiment, such as samples without one of the fluorescent proteins, to distinguish specific signals from non-specific bleed-through. This can help validate the specificity of the detected signals.

By implementing these optimization strategies, the bleed-through of signals when visualizing green- and red-tagged proteins in the same sample can be minimized, enhancing the accuracy and reliability of IGF detection in multi-color experiments.

How could the IGF detection method be further optimized to minimize the bleed-through of signals when visualizing green- and red-tagged proteins in the same sample?

The IGF detection approach can be extended to study a wide range of post-translational modifications (PTMs) and protein interactions beyond co-immunoprecipitation experiments. Some potential applications include:

Phosphorylation Studies: IGF can be used to investigate phosphorylation events by tagging proteins with FPs and analyzing changes in fluorescence intensity or migration patterns on gels in response to phosphorylation.

Ubiquitination and SUMOylation: By tagging proteins involved in ubiquitination or SUMOylation pathways with FPs, IGF can be utilized to study the dynamics of these PTMs and their impact on protein stability and function.

Acetylation and Methylation: IGF detection can be applied to study protein acetylation and methylation events by visualizing changes in fluorescence patterns or mobility shifts on gels upon these modifications.

Protein-Protein Interactions: Beyond co-immunoprecipitation, IGF can be used to investigate protein-protein interactions by tagging interacting partners with FPs and analyzing changes in fluorescence signals upon complex formation.

Subcellular Localization Studies: IGF can aid in studying protein localization dynamics by monitoring the fluorescence of tagged proteins in different cellular compartments, providing insights into organelle targeting and trafficking.

Proteolytic Cleavage Events: IGF detection can be employed to study proteolytic cleavage events by monitoring changes in fluorescence intensity or migration patterns of proteins undergoing cleavage.

By leveraging the versatility and sensitivity of IGF detection, researchers can explore a wide array of PTMs and protein interactions, offering valuable insights into the dynamic regulation of cellular processes.

Given the versatility of the IGF detection method, how could it be adapted or combined with other techniques to enable new types of biological investigations or expand the range of applications in cell and molecular biology research?

The versatility of the IGF detection method opens up opportunities for innovative biological investigations and applications in cell and molecular biology. Here are some ways it could be adapted or combined with other techniques to enable new types of research:

Single-Cell Analysis: Integration of IGF with single-cell analysis techniques, such as single-cell RNA sequencing or mass cytometry, can provide spatial and functional information about protein expression and modifications at the single-cell level.

High-Throughput Screening: Combining IGF with high-throughput screening platforms can facilitate large-scale studies of protein interactions, PTMs, and localization dynamics, accelerating drug discovery and functional genomics research.

Live-Cell Imaging: Integration of IGF with live-cell imaging techniques, such as fluorescence microscopy or super-resolution imaging, can enable real-time visualization of protein dynamics and interactions in living cells.

Quantitative Proteomics: IGF can be coupled with quantitative proteomics approaches, such as mass spectrometry-based proteomics, to validate protein expression levels and modifications identified through mass spectrometry analysis.

Structural Biology: Integration of IGF with structural biology techniques, such as X-ray crystallography or cryo-electron microscopy, can provide complementary information on protein structure and conformational changes.

Functional Genomics: Combining IGF with CRISPR-based genetic screens or RNA interference studies can elucidate the functional roles of proteins and their modifications in cellular processes.

Multi-Omics Integration: Integration of IGF with other omics techniques, such as transcriptomics and metabolomics, can provide a comprehensive view of cellular processes and regulatory networks.

By adapting and integrating IGF with other cutting-edge techniques, researchers can explore new avenues of biological investigation, expand the range of applications in cell and molecular biology research, and gain deeper insights into the complex mechanisms governing cellular function and disease processes.