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November 15, 2011 (Vol. 31, No. 20)

Stain-Free Approach for Western Blotting

Alternative to the Standard Blot Normalization Process

  • Protein-Profiling Studies

    Click Image To Enlarge +
    Figure 2a. LCL cell lysates (30 µg total protein per lane) separated by TGX and kD Criterion Stain-Free 1-D SDS gel, blotted to PVDF membrane and imaged with ChemiDoc MP.

    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.

  • Click Image To Enlarge +
    Figure 2b. Immunofluorescence imaging of DNA replication factor MCM7 and housekeeping protein GAPDH (serves as loading control) of LCL samples with ChemiDoc MP at different wavelengths. Monoclonal antibodies against MCM7 (mouse) and GAPDH (rabbit) were diluted 1:1,000 and 1:2,500 respectively. Secondary antibodies from Rockland were antirabbit DyLight 549 (1:10,000) and antimouse DyLight 649 (1:20,000).

    Figure 2b illustrates probing the same PVDF blot for target protein MCM7 and loading control protein GAPDH using a multiplex fluorescent approach. By running a dilution series of the LCL samples from 10–50 µg per lane, it was verified beforehand that the 30 µg protein load in combination with the antibody dilutions as described in Figure 2B give linear fluorescent signals.

    Data normalization of the MCM7 signal intensities with GAPDH data and Stain-Free data revealed that the downregulation factors of MCM7 are 0.59 with GAPDH (p-value <0.03) and 0.44 with Stain-Free (p-value <0.008). Both factors reflect well with the expected downregulation of about 50% initially obtained from 2-D PAGE experiments. The comparison of raw MCM7 signals (no normalization) reveals a downregulation factor of 0.48 (p-value <0.02) with 35% standard deviation.

    Although it is possible to use housekeeping proteins as reference for data normalization, especially if used in a multiplex fluorescent experiment as described here, it is highly recommended to consider Stain-Free technology as a more comprehensive and robust tool for quality management in Western blotting, particularly when protein deregulation factors are small, e.g., in the 10% to 20% range.

  • ChemiDoc MP Imaging System

    Bio-Rad’s latest imaging system, the ChemiDoc MP, is designed to enhance Western blotting. As Bio-Rad’s most sensitive imager, the ChemiDoc MP is designed to meet the imaging needs of a multi-application environment. Capabilities include chemiluminescent and multiplex fluorescent Western blot detection, and gel documentation.

    The Stain-Free feature of ChemiDoc MP enables researchers to take advantage of techniques such as Stain-Free total protein normalization. Whether users are accustomed to chemiluminescent detection, multiplex fluorescent detection, or would like to take advantage of Stain-Free technology, the versatility of the ChemiDoc MP meets the diverse needs of modern laboratories.

  • Conclusion

    The use of Bio-Rad Stain-Free gels and a Stain-Free enabled imaging system can vastly improve the Western blot process. Protein separation can be visualized on the gel after electrophoresis. Protein transfer to the membrane can be confirmed and assessed prior to blot detection, and blot normalization can occur without the extra steps required for typical HKP detection. These improvements enable faster time-to-results and higher data confidence.

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