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Application of glutaraldehyde to in-cell Western assay for normalization

Jingbao Li1, Zhe Wang, Gang He

  • 1Key Laboratory for Space Bioscience and Biotechnology, Faculty of Life Sciences, Northwestern Polytechnical University, Xi'an, China.

Analytical Biochemistry
|December 19, 2009
PubMed

Insights

Glutaraldehyde treatment enables near-infrared fluorescence for cell quantification in the in-cell Western assay. This simplifies normalization, potentially increasing the assay's popularity.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Immunotechnology

Background:

  • The in-cell Western (ICW) assay is a crucial method for protein analysis using near-infrared immunocytoblotting.
  • Accurate normalization is vital for reliable protein quantification in ICW assays.
  • Existing normalization methods can be complex or costly.

Purpose of the Study:

  • To develop a novel, simplified method for cell quantification to normalize in-cell Western assays.
  • To leverage glutaraldehyde-induced near-infrared fluorescence for cell counting.

Main Methods:

  • Cells were treated with glutaraldehyde.
  • Near-infrared fluorescence intensity was measured.
  • Fluorescence intensity was correlated with cell number (3125–100,000 cells/well).
  • The glutaraldehyde method was applied for normalization in ICW assays.

Main Results:

  • Glutaraldehyde treatment induced near-infrared fluorescence in cells.
  • Fluorescence intensity showed a direct, proportional relationship with cell number.
  • The method provided accurate cell quantification for ICW assay normalization.

Conclusions:

  • Glutaraldehyde treatment offers a straightforward and cost-effective method for cell normalization in ICW assays.
  • This approach simplifies the ICW assay procedure.
  • The use of glutaraldehyde may enhance the adoption and popularity of the ICW assay.

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