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In-Cell ELISA uses whole cells, allowing easy conversion to high throughput analysis e.g. for drug screening.
(optional) Whole cell staining with Janus Green (a mitochondrial dye) to normalize to cell number
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.
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.
MitoBiogenesis™ In-Cell ELISA Kit (ab110216) can also be used to determine IC50 of a drug on mitochondrial protein translation.
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.
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.
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).