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An Automated Reinvention of the Western Blot ‐ A Simple Western Analysis of the AKT Pathway Signaling Cascade
Author(s) -
Boge Annegret,
Ramirez Francisco,
Nguyen Uyen,
Kazakova Irina,
Dermody Jessica,
Yang Tom,
Gavin Robert
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.776.13
Subject(s) - western blot , protein kinase b , blot , proteome , microbiology and biotechnology , cascade , signal transduction , chemistry , biology , biochemistry , chromatography , gene
Protein kinase AKT is a central player for a variety of cell stimuli affecting downstream events such as the inhibition of apoptosis, regulation of glycogen synthesis, and cell cycle regulation. Many of these events are routinely assessed by Western blot analysis which exploits high sensitivity of enzyme amplification as well as specificity of antibody binding. The process itself, however, is very labor intensive and has not significantly changed since it was first introduced. Simple Western assays utilize capillary electrophoresis, a powerful separation technique with high resolution and reproducibility, and incorporate sample separation performed in capillaries followed by immobilization of the proteins directly onto the capillary walls, followed by immunodetection. Data comparing analysis of key targets of the AKT signaling cascade via Western blot and Simple Western highlighting workflow, biological response, sensitivity, and resolution is presented. Conclusions Simple Western assays are a reinvention of the Western blot, automating all the manual steps associated with the process. Highly reproducible, quantitative, reliable and sensitive assessments of key targets in the AKT signaling cascade were observed. Additionally, total time to results was dramatically pared down to 3–5 hours in a walk‐away mode with a total hands‐on time of less than an hour.