What Is In-Cell ELISA? The Modified Procedure for a More Accurate Assay

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.


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


Leave your vote

-1 points
Upvote Downvote

Leave a Reply

Your email address will not be published.

Log In

Forgot password?

Forgot password?

Enter your account data and we will send you a link to reset your password.

Your password reset link appears to be invalid or expired.

Log in

Privacy Policy

Add to Collection

No Collections

Here you'll find all collections you've created before.