An analysis of critical factors for quantitative immunoblotting

被引：163

作者：

Janes, Kevin A. ^{[1
]}

机构：

[1] Univ Virginia, Dept Biomed Engn, Charlottesville, VA 22908 USA

来源：

SCIENCE SIGNALING | 2015年 / 8卷 / 371期

关键词：

BREAST EPITHELIAL-CELLS; REGULATORY HETEROGENEITIES; SIGNALING NETWORKS; PROTEIN; ASSAY; NITROCELLULOSE; FLUORESCENCE; SEPARATION; INHIBITION; PROGRAMS;

D O I：

10.1126/scisignal.2005966

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Immunoblotting (also known as Western blotting) combined with digital image analysis can be a reliable method for analyzing the abundance of proteins and protein modifications, but not every immunoblot-analysis combination produces an accurate result. I illustrate how sample preparation, protocol implementation, detection scheme, and normalization approach profoundly affect the quantitative performance of immunoblotting. This study implemented diagnostic experiments that assess an immunoblot-analysis workflow for accuracy and precision. The results showed that ignoring such diagnostics can lead to pseudoquantitative immunoblot data that markedly overestimate or underestimate true differences in protein abundance.

引用

页数：11

共 48 条

[41] A Defined Methodology for Reliable Quantification of Western Blot Data [J].

Taylor, Sean C. ;

Berkelman, Thomas ;

Yadav, Geetha ;

Hammond, Matt .

MOLECULAR BIOTECHNOLOGY, 2013, 55 (03) :217-226

[42] ELECTROPHORETIC TRANSFER OF PROTEINS FROM POLYACRYLAMIDE GELS TO NITROCELLULOSE SHEETS - PROCEDURE AND SOME APPLICATIONS [J].

TOWBIN, H ;

STAEHELIN, T ;

GORDON, J .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1979, 76 (09) :4350-4354

[43] MONOCLONAL-ANTIBODY - MEDIATED TUMOR-REGRESSION BY INDUCTION OF APOPTOSIS [J].

TRAUTH, BC ;

KLAS, C ;

PETERS, AMJ ;

MATZKU, S ;

MOLLER, P ;

FALK, W ;

DEBATIN, KM ;

KRAMMER, PH .

SCIENCE, 1989, 245 (4915) :301-305

[44] Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies [J].

Tricarico, C ;

Pinzani, P ;

Bianchi, S ;

Paglierani, M ;

Distante, V ;

Pazzagli, M ;

Bustin, SA ;

Orlando, C .

ANALYTICAL BIOCHEMISTRY, 2002, 309 (02) :293-300

[45] Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes [J].

Vandesompele, Jo ;

De Preter, Katleen ;

Pattyn, Filip ;

Poppe, Bruce ;

Van Roy, Nadine ;

De Paepe, Anne ;

Speleman, Frank .

GENOME BIOLOGY, 2002, 3 (07)

[46] A time-and matrix-dependent TGFBR3-JUND-KRT5 regulatory circuit in single breast epithelial cells and basal-like premalignancies [J].

Wang, Chun-Chao ;

Bajikar, Sameer S. ;

Jamal, Leen ;

Atkins, Kristen A. ;

Janes, Kevin A. .

NATURE CELL BIOLOGY, 2014, 16 (04) :345-+

[47] Stochastic profiling of transcriptional regulatory heterogeneities in tissues, tumors and cultured cells [J].

Wang, Lixin ;

Janes, Kevin A. .

NATURE PROTOCOLS, 2013, 8 (02) :282-301

[48] Intersection of FOXO- and RUNX1-mediated gene expression programs in single breast epithelial cells during morphogenesis and tumor progression [J].

Wang, Lixin ;

Brugge, Joan S. ;

Janes, Kevin A. .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2011, 108 (40) :E803-E812

← 1 2 3 4 5 →