Proteomic technologies like 2-D PAGE are customarily used in semi-quantitative protein-profiling studies. After identification and characterization of the respective deregulated protein spots by mass spectrometry, the quantitative data needs to be confirmed by a second, independent method like Western blotting.
In a recent proteomics study conducted by the department of radiation oncology at LMU, Munich, the effect of γ-irradiation on a human LCL cell line (lymphoblastoid cell line) was studied by 2-D PAGE. Among many other deregulated proteins, MCM7, a DNA replication factor, was identified to be down-regulated by about a factor of two in the irradiated LCL sample.
To validate this result, a quantitative Western blotting experiment was performed with monoclonal antibodies raised against MCM7. Two data-normalization methods were applied and compared, namely data normalization with the GAPDH HKP and Stain-Free technology.
Figure 2A shows the corresponding high-quality PVDF blot of the LCL samples before antibody incubation.