All tags In-Cell ELISA Get quantitative data from In-Cell ELISA

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Get quantitative data from In-Cell ELISA

In-Cell ELISA is a specific and quantitative technique for the analysis of your target of interest, and uses the ELISA method for detection.

In-Cell ELISA uses whole cells, allowing easy conversion to high throughput analysis e.g. for drug screening.

There are just 6 basic steps in the In-Cell ELISA:

  1. Prepare cells in microplate
  2. Fix cells to microplate
  3. Permeabilize cells
  4. Incubate with primary antibody
  5. Incubate with secondary antibody
  6. Measure signal (colorimetric, fluorometric or infrared)

​​​​(optional) Whole cell staining with Janus Green (a mitochondrial dye) to normalize to cell number

In-Cell ELISA diagram

Watch our video to see how an In-Cell ELISA works.  

Microplate signal measurement

Some of our In-Cell ELISA kits use AP or HRP-labeled secondary antibodies, and can be read on a standard microplate reader. Kits using IRDye®-labeled secondary antibodies can be read with a LiCor® Odyssey® or Aerius® scanner. Duplex readouts from a single well can be obtained if using IR-800 and IR-680 labeled secondary antibodies.  

Here are a few examples showing how you can use data from In-Cell ELISAs:

Measuring changes to mitochondrial and nuclear DNA encoded proteins

Our MitoBiogenesis™ In-Cell ELISA kit (ab110216) detects specific inhibition of mitochondrial encoded protein COX-1 (MTCO1) and nuclear protein SDH-A.

​​​​​Rat H9C2 cardiomyocytes are seeded at between 100,000 cells/well (top row of plate) and 1,000 cells/well (bottom row of plate). Chloramphenicol is then added to the cells. After several doublings of cells, the plate is read.​​​​​​​​​​​

ab110216 in cell elisa figure 1

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​​​​Figure 1  ​Specific inhibition of mitochondrial encoded protein synthesis detected. Using MitoBiogenesis™​ In-Cell ELISA Kit  (ab110216), mitochondrial encoded protein COX-1 (MTCO1) is detected at 800 nm (green) and nuclear encoded protein SDH-A at 700 nm (red). The merged image shows the relative ratio of MTCO1 (COX-1) and SDH-A proteins. A yellow signal indicates normal levels of both proteins (as both signals are approximately equal). An orange signal indicates low levels of MTCO1 (COX-1) and normal levels of SDH-A. Figure 1b shows results are independent of cell number, once signal is adjusted for cell seeding density.

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​​​​​​MitoBiogenesis™​ In-Cell ELISA Kit (ab110216) can also be used to determine IC50 of a drug on mitochondrial protein translation. 

ab110216 in cell elisa ic50

​​​​​Figure 2:  IC50 of drug on mitochondrial protein translation calculated. Rat H9C2 cardiomyocytes are seeded at 3,000 cells/well and are allowed to grow for 3 cell doublings in a drug dilution series. Using MitoBiogenesis™In-Cell ELISA Kit (ab110216), relative amounts of MTCO1 (COX-I) and SDH-A are measured in each well. Chloramphenicol inhibits mitochondrial MTCO1 (COX-I) protein synthesis relative to nuclear DNA-encoded SDH-A protein synthesis by 50% at 3.5 µM.

Measuring changes in phosphorylated intracellular proteins

​​​​​​STAT5 (Tyr694) Human In-Cell ELISA Kit (ab126429) ​ can be used to measure relative amount of STAT5 (Tyr694) phosphorylation as a results of treatment with e.g., inhibitors (such as siRNA or chemicals), or activators in cultured human cell lines. 


analysis
ab126429 STAT5 (Tyr 694) in cell ELISA

Figure 3: Increased levels of  STAT5 phosphorylation detected. Using STAT5 (Tyr694) Human In-Cell ELISA Kit (ab126429) A341 cells (20,000 cells/well) are seeded and incubated overnight at 37°C in 5% CO2. Different concentrations of recombinant human EGF (rhEGF) in serum-free DMEM are added to the cells and incubated for 30 minutes at 37°​C.  Increasing levels of STAT5 phosphorylation are detected with increasing concentrations of EGF.

​​H2A.X (pSer139) Human In-Cell ELISA Kit (ab131382) measures H2A.X serine 139 phosphorylation, an indicator of DNA damage.

ab131382 In-Cell ELISA H2A.X phosphorylation

​​​​Figure 4: Increased levels of DNA damage detected. HeLa cells were treated for 4 hours with titrated doses of camptothecin, etoposide and staurosporine. With increasing concentrations of drug, increasing levels of H2A.X pSer139 are detected using H2A.X (pSer139) Human In-Cell ELISA Kit (ab131382). The dashed grey line indicates vehicle control signal. Signal is normalized to account for cell seeding density, using Janus Green (a mitochondrial dye).



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