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Antibody Validation By Western Blotting

fig1WesternBlotting

Figure 1. Specificity of PARP-1 (cleaved) antibody demonstrated by Western blot. Western blot analysis of HeLa cells either untreated (CON) or treated with 1 μM Staurosporine (STS) for 4 hours to induce apoptosis. This antibody recognizes cleaved PARP-1 (89 kDa) and not full length PARP-1 (113 kDa). Antibody was from MitoSciences PARP-1 (cleaved) In-Cell ELISA Kit (IR) (ab110215).

Western blots yield useful information about specificity, even though antigen presentation is different than in fixed cells.1,2

  • Confirm correct molecular weights of target protein (Fig. 1)
    • NOTE: Some primary antibodies may not recognize denatured antigens on Western blots.
    • Western blot specificity does not guarantee specificity in fixed cells.
    • An antibody with strong non-specific bands may not be suitable for ICW assays
  • Confirm that desired cellular response is triggered in your experiments (Fig. 2)
  • Optimize cell treatment conditions and timing to capture the peak response
    • Phosphorylation levels may increase and decrease rapidly. Sampling at the wrong time may overlook or underestimate the response.
fig2WesternBlotting

Figure 2. Inhibition of mitochondrial biogenesis by chloramphenicol. A) Western blot validation. Chloramphenicol treatment inhibits mtDNA-encoded COX-1 protein synthesis, but not nuclear-DNA-encoded SDH-A protein synthesis. B) The IC50 of the drug's effect on mitochondrial protein translation was determined with the Mitosciences MitoBiogenesis™ In-Cell ELISA Kit (IR) (ab110216). Cells were seeded at 3000 cells/well and allowed to grow for 3 cell doublings in a drug dilution series. Relative amounts of COX-1 and SDH-A were measured in each well. Chloramphenicol inhibits COX-1 protein synthesis relative to SDH-A protein synthesis by 50% at 3.5 μM.

  • 1. Aguilar HN et al. PLoS ONE 5(4): e9965 (2010)
  • 2. In-Cell Western assay primary antibody selection and validation Application Note LI-COR (2011)


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