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Protein Expression

Quantify fluorescence intensity on a cell by cell basis using nuclear and cellular markers

One of the most common assays in cell biology is the protein expression.

This is a very basic tool that can be applied for numerous assays, such as dose response assays, protein degradation, Transfection efficiency and many more.

  • Quantify fluorescence intensity on a cell-by-cell basis using nuclear and/or cytoplasmic markers.
  • Cells in each image are automatically identified and their fluorescence intensity measured.  A heat-map overlaying the plate layout (top) presents the comparative results for intuitive data visualization.
  • Athena’s fast and labor reducing  enables users to rapidly screen a large variety of experimental conditions, as with transfection for example, that uses comparative protein expression as a readout.
  • Enables simultaneous studies of mixed populations

 

Assay Groups- What is it good for?

  • Cell Health / Autophagy assays
  • Protein Localization & translocation
  • Subcellular distribution analysis
  • Protein degradation/ Accumulation assays (t1/2) / Ubiquitination

 

 

  • Dose response assays
  • Transfection efficiency assay
  • Gene therapy assays
  • Visualize Response to therapies & drug mechanisms

Autophagy Assay

Protein Expression Athena application applied for detection of cell viability (Autophagy), protein expression and protein phosphorylation Assay in breast cancer research:

Breast cancer drug neratinib has been found to block the function of the Ras genes which are among the first to be linked to cancer development.

Image analysis showed Neratinib down-regulating the expression of mutant K-RAS and mutant N-RAS.

Neratinib and HDAC inhibitors interact to reduce the expression of mutant N-RAS and mutant K-RAS. Spiky and PANC1 cells that express a mutant N-RAS and a mutant K-RAS, respectively, were treated with vehicle control and neratinib (50 nM). The cells were fixed in place and immunostaining performed with DAPI counter-stain to determine the expression levels and cellular localization of N-RAS and K-RAS at 60× magnification using Hermes HCS system.

Source: 

[Neratinib + Valproate] exposure permanently reduces ERBB1 and RAS expression in 4T1 mammary tumors and enhances M1 macrophage infiltration. Booth et al., Oncotarget, 26;9(5):6062-6074Dec 2017  WEB LINK

Transfection Efficiency Assay

Hermes enables users to efficiently study a large variety of conditions for transfection and enables simultaneous studies of many cell lines.

Results shown present a heat map of 6 cell lines (3×3 wells shown in white squares) studied for transfection efficiency.

The upper 6 wells are transfected.

The lower 3 wells are untransfected control.

Images were acquired by Hermes using  with magnification 10X and were analyzed using the Athena Protein Expression application.

 

“In-Cell Westerns” Assay

Assessments of total protein expression or post translational modifications, such as phosphorylation, can be made via fluorescence intensity measurements.  Such “in-cell western” analysis uses unbiased pre-programmed electronic data acquisition, much as has previously been performed for the last 45 years using SDS-PAGE and western immunoblotting.

Source:                                  

Rationally repurposing Ruxolitinib (Jakafi®) as a solid tumor therapeuticTavallai Mehrad, Booth Laurence, Roberts Jane L, Poklepovic Andrew, Dent Paul ; Frontiers in Oncology ,vol.6,2016  WEB LINK

 App note-A novel approach to detect gross changes in protein expression and protein phosphorylation

The table above compares main differences between the conventional western blot assay and protein expression endearments using the Hermes high content imaging system. 

 

For more information please contact us at info@idea-bio.com