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Intracellular Protein Detection

NovoCyte Penteon Flow Cytometer

Detection and analysis of intracellular proteins allow for additional characterization of cell subpopulations and cellular processes. In order to analyze proteins not located on the cell surface, fixation and permeabilization of the cell is required. However, many phospho-specific antibodies are not compatible with many common detergent-based permeabilization methods used for intracellular staining. Special attention is needed when determining the proper fix/perm method for your phospho-specific antibody. The most common method uses 1.5% paraformaldehyde for fixation followed by 100% methanol for permeabilization. While this method works for many antibodies, please note it may not work for every phospho-specific antibody.

Additionally, identifying various cell populations in a heterogenous sample requires staining for phosphorylated proteins coupled with surface proteins. Special consideration must be given to the sensitivity of these epitopes to fixative, taking precaution to avoid damage to the epitope. Therefore, the sample may require staining for specific surface markers before fixation.

Flow cytometry has traditionally been used to detect extracellular proteins to identify different cell populations. With the advancement of technology and reagents in recent years, we now are able to easily detect intracellular proteins in individual cells. One very useful application for the detection of intracellular proteins is to measure specific cells signaling pathways. These pathways are often altered in disease states, such as cancer, and are now easily detected using flow cytometry providing single cell analysis of these signaling changes.

NovoCyte Penteon Flow Cytometer

Jurkat cells were either left in culture(pERK unstim) or stimulated with cell stimulation cocktail for 1hr (pERK +stim).  Cells were washed, fixed and permeablized, followed by staining with anti-phospo-ERK1/2 (T204/Y202) PerCP antibody for 1hr.  Cells were washed and analyzed on the NovoCyte flow cytometer for phospho-ERK levels and compared using a histogram overlay generated with the NovoExpress acquisition and analysis software.

NovoCyte Penteon Flow Cytometer


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