title: Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system.
abstract: Precise and accurate quantification of protein expression levels in a complex biological setting is challenging. Here, we describe a method for absolute quantitation of endogenous proteins in cell lysates using an automated capillary immunoassay system, the size-based Simple Western system (recently developed by ProteinSimple). The method was able to accurately measure the absolute amounts of target proteins at picogram or sub-picogram levels per nanogram of cell lysates. The measurements were independent of the cell matrix or the cell lysis buffer and were not affected by different antibody affinities for their specific epitopes. We then applied this method to quantitate absolute levels of expression of protein kinase C (PKC) isoforms in LNCaP and U937 cells, two cell lines used extensively for probing the downstream biological responses to PKC targeted ligands. Our absolute quantitation confirmed the predominance of PKCδ in both cells, supporting the important functional role of this PKC isoform in these cell lines. The method described here provides an approach to accurately quantitate levels of protein expression and correlate protein level with function. In addition to enhanced accuracy relative to conventional Western analysis, it circumvents the distortions inherent in comparison with signal intensities from different antibodies with different affinities.
authors.affiliation: Collaborative Protein Technology Resource, Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: chenj13@mail.nih.gov.
authors.forename: Jin-Qiu
authors.initials: JQ
authors.lastname: Chen
authors.forename: Madeleine R
authors.initials: MR
authors.lastname: Heldman
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Herrmann
authors.forename: Noemi
authors.initials: N
authors.lastname: Kedei
authors.forename: Wonhee
authors.initials: W
authors.lastname: Woo
authors.forename: Peter M
authors.initials: PM
authors.lastname: Blumberg
authors.forename: Paul K
authors.initials: PK
authors.lastname: Goldsmith
mesh_terms: D001331:Automation; D015153:Blotting, Western; D045744:Cell Line, Tumor; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D011493:Protein Kinase C / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D011493:Protein Kinase C
keywords: Capillary immunoassay; Protein kinase C (PKC); Quantitative protein analysis; Simple Western
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