In-Cell ELISA is a highly regarded quantitative method for detecting and measuring proteins in cells. This immunocytochemistry assay determines the concentration of a protein in a cell lysate or culture supernatant, according to abcam.com. The principle behind In-Cell ELISA is that soluble proteins will bind to an antibody labeled with a fluorescent molecule. The assay can perform in either adherent or suspension cells. Other standard terms for In-Cell ELISA are Cell-Based ELISA, In-Cell-Western (ICW) ELISA, or cytobot. The In-Cell ELISA procedure is similar to a traditional Western blot assay, with a few key differences that can make it more accurate.
In-Cell ELISA Procedure
The In-Cell ELISA procedure aims to extract proteins from cells and detect the presence of a target protein. The basic procedure for performing In-Cell ELISA is as follows:
- Dilute the cell lysate or culture supernatant in a buffer that does not contain detergents.
- Add the diluted sample to a microplate well that has been coated with an antibody against the protein of interest.
- Incubate the plate at standard room temperature for two hours.
- Remove the unbound antibodies by washing the plate three times with a buffer that does not contain detergents.
- Add a second antibody labeled with a fluorescent molecule to each well.
- Incubate the plate again at room temperature for two hours.
- Remove the unbound antibodies by washing the plate three times with a buffer that does not contain detergents.
- Count the number of fluorescently labeled antibodies in each well using a fluorescence-activated cell sorting (FACS) machine.
In-Cell ELISA Benefits
Several factors should be considered when deciding what assay to use. Western Blot is a commonly used assay for detecting and measuring proteins in cells. However, In-Cell ELISA has several advantages over Western Blot: it is more sensitive, specific, and quantitative.
The benefits of using In-Cell ELISA over Western Blot include:
- Increased sensitivity: The assay is more sensitive than Western Blot and can detect proteins at lower concentrations depending on the specific signature used for measurement according to Future-Science.
- Increased specificity: The assay is more specific than Western Blot and can distinguish between closely related proteins.
- Heightened quantitative accuracy: The assay is more quantitative than Western Blot and can provide more accurate protein concentration measurements.
- Characterize a broader range of cell signaling parameters: Western Blot is limited to measuring the phosphorylation state of proteins. In-Cell ELISA can also measure protein localization, interaction, and degradation.
- More effective protein analysis: The assay can be performed in adherent or suspension cells, allowing for the study of both soluble and insoluble proteins.
- Ease of performance: The assay is relatively easy to perform and does not require specialized equipment or expertise.
- Concentration insight: The assay can be used to measure the concentration of proteins in cell lysates or culture supernatants.
- Shorter protocol: The assay has a shorter protocol than Western Blot and can be completed in a few hours due to shorter incubation times, faster washes, and the lack of electrophoresis.
- Reduced variability: The assay is less variable than Western Blot due to the smaller standard of deviations that are typically observed.
- Replicate efficient measurements: The assay can replicate measurements at a very low level of coefficients of variation. This allows for the accurate determination of protein concentrations in addition to processing many samples or replicates to have a more streamlined workflow.
Using In-Cell ELISA over Western Blot provides a more accurate and quantitative assay for detecting proteins in cells. The ease of performance and shorter protocol makes it a more convenient assay. In-Cell ELISA is becoming the assay of choice for protein analysis and should be considered when designing a protein detection assay.
Sources:
https://www.frontiersin.org/articles/10.3389/fimmu.2020.573526/full https://www.future-science.com/doi/epdf/10.2144/03354ss02 https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3877136 https://www.abcam.com/protocols/in-cell-elisa-ice
