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  • Capillary electrophoresis W...
    Booth, Peter-Philip M.; Lamb, Don T.; Anderson, Jon P.; Furtaw, Michael D.; Kennedy, Robert T.

    Journal of Chromatography A, 08/2022, Letnik: 1679
    Journal Article

    •Western blotting is performed from nanogram scale samples.•Protein samples are separated and captured on a membrane in 15 minutes.•Fluorescent antibodies are used to simultaneously detect ladder and analyte. Traditional Western blots are commonly used to separate and assay proteins; however, they have limitations including a long, cumbersome process and large sample requirements. Here, we describe a system for Western blotting where capillary gel electrophoresis is used to separate sodium dodecyl sulfate-protein complexes. The capillary outlet is threaded into a piezoelectric inkjetting head that deposits the separated proteins in a quasi-continuous stream of <100 pL droplets onto a moving membrane. Through separations at 400 V/cm and protein capture on a membrane moving at 2 mm/min, we are able to detect actin with a limit of detection at 8 pM, or an estimated 5 fg injected. Separation and membrane capture of sample containing 10 proteins ranging in molecular weights from 11 – 250 kDa was achieved in 15 min. The system was demonstrated with Western blots for actin, β-tubulin, ERK1/2, and STAT3 in human A431 epidermoid carcinoma cell lysate.