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A fully automated capillary western system for absolute quantitation of endogenous PKC proteins: An approach to correlate protein quantity with function.

Publication ,  Conference
Chen, J-Q; Heldman, M; Herrmann, M; Kedei, N; Blumberg, P; Goldsmith, P
Published in: Journal of Clinical Oncology
October 20, 2012

31 Background: The human prostate cell line LNCaP and the human myelocytic leukemia cell line U937 differ dramatically in their responses to the two protein kinase C (PKC) targeted ligands phorbol 12-myristate 13-acetate (PMA) and bryostatin 1 and show complex differences in the patterns of transcriptional responses that they induce. Quantitation of relative abundance of individual PKC isoforms in the two cell lines may help to link the downstream effects of the two compounds to these isoforms. Methods: Simple Western is a capillary-based automated Western system recently developed by ProteinSimple. All steps following sample preparation are fully automated in the Simple Western system, including sample loading, size-based protein separation, immunoprobing, washing, detection and data analysis. Simple Western is gel-free and blot-free, uses less amount of samples, and produces highly quantitative, reproducible information that cannot be generated using regular Western assays. Using the Simple Western system, we developed a method for absolute quantitation of endogenous proteins in cell lysates and quantified PKC isoforms in LNCaP and U937 cells. Results: PKC isoforms were measured at levels of picogram or sub-picogram per nanogram cell lysate. PKC delta was identified as the dominant PKC isoforms in both cell lines. In LNCaP cells, PKC delta expression is ~20-fold higher than PKC alpha, ~40-fold higher than PKC epsilon, and at least 20-fold higher than PKC beta. In U937 cells, PKC delta expression is similar to PKC beta, at least 200 fold higher than PKC alpha, and ~50-fold higher than PKC epsilon. Conclusions: The Simple Western system, with its high-quality data quantitation and excellent assay reproducibility, allowed us to detect both the relative abundance of the PKC isoforms and their absolute quantitation in the tested cells. It circumvents the problem that antibodies of different affinities for different proteins yield a misleading impression of relative abundance and it provides an approach to accurately correlate protein quantities with their function.

Duke Scholars

Published In

Journal of Clinical Oncology

DOI

EISSN

1527-7755

ISSN

0732-183X

Publication Date

October 20, 2012

Volume

30

Issue

30_suppl

Start / End Page

31 / 31

Publisher

American Society of Clinical Oncology (ASCO)

Related Subject Headings

  • Oncology & Carcinogenesis
  • 3211 Oncology and carcinogenesis
  • 1112 Oncology and Carcinogenesis
  • 1103 Clinical Sciences
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Chen, J.-Q., Heldman, M., Herrmann, M., Kedei, N., Blumberg, P., & Goldsmith, P. (2012). A fully automated capillary western system for absolute quantitation of endogenous PKC proteins: An approach to correlate protein quantity with function. In Journal of Clinical Oncology (Vol. 30, pp. 31–31). American Society of Clinical Oncology (ASCO). https://doi.org/10.1200/jco.2012.30.30_suppl.31
Chen, Jin-Qiu, Madeleine Heldman, Michelle Herrmann, Noemi Kedei, Peter Blumberg, and Paul Goldsmith. “A fully automated capillary western system for absolute quantitation of endogenous PKC proteins: An approach to correlate protein quantity with function.” In Journal of Clinical Oncology, 30:31–31. American Society of Clinical Oncology (ASCO), 2012. https://doi.org/10.1200/jco.2012.30.30_suppl.31.
Chen J-Q, Heldman M, Herrmann M, Kedei N, Blumberg P, Goldsmith P. A fully automated capillary western system for absolute quantitation of endogenous PKC proteins: An approach to correlate protein quantity with function. In: Journal of Clinical Oncology. American Society of Clinical Oncology (ASCO); 2012. p. 31–31.
Chen, Jin-Qiu, et al. “A fully automated capillary western system for absolute quantitation of endogenous PKC proteins: An approach to correlate protein quantity with function.Journal of Clinical Oncology, vol. 30, no. 30_suppl, American Society of Clinical Oncology (ASCO), 2012, pp. 31–31. Crossref, doi:10.1200/jco.2012.30.30_suppl.31.
Chen J-Q, Heldman M, Herrmann M, Kedei N, Blumberg P, Goldsmith P. A fully automated capillary western system for absolute quantitation of endogenous PKC proteins: An approach to correlate protein quantity with function. Journal of Clinical Oncology. American Society of Clinical Oncology (ASCO); 2012. p. 31–31.

Published In

Journal of Clinical Oncology

DOI

EISSN

1527-7755

ISSN

0732-183X

Publication Date

October 20, 2012

Volume

30

Issue

30_suppl

Start / End Page

31 / 31

Publisher

American Society of Clinical Oncology (ASCO)

Related Subject Headings

  • Oncology & Carcinogenesis
  • 3211 Oncology and carcinogenesis
  • 1112 Oncology and Carcinogenesis
  • 1103 Clinical Sciences