Quantification of rapid Myosin regulatory light chain phosphorylation using high-throughput in-cell Western assays: comparison to Western immunoblots.

Aguilar HN, Zielnik B, Tracey CN, Mitchell BF

PloS one1 Apr 2010Cited by 44 (Europe PMC)

PMID20376358PMCIDPMC2848601DOI10.1371/journal.pone.0009965PepBankPUB-20376358

Summary

BackgroundQuantification of phospho-proteins (PPs) is crucial when studying cellular signaling pathways. Western immunoblotting (WB) is commonly used for the measurement of relative levels of signaling intermediates in experimental samples. However, WB is in general a labour-intensive and low-throughput technique. Because of variability in protein yield and phospho-signal preservation during protein harvesting, and potential loss of antigen during protein transfer, WB provides only semi-quantitative data. By comparison, the "in-cell western" (ICW) technique has high-throughput capacity and requires less extensive sample preparation. Thus, we compared the ICW technique to WB for measuring phosphorylated myosin regulatory light chain (PMLC(20)) in primary cultures of uterine myocytes to assess their relative specificity, sensitivity, precision, and quantification of biologically relevant responses.Methodology/principal findingsICWs are cell-based microplate assays for quantification of protein targets in their cellular context. ICWs utilize a two-channel infrared (IR) scanner (Odyssey(R)) to quantify signals arising from near-infrared (NIR) fluorophores conjugated to secondary antibodies. One channel is dedicated to measuring the protein of interest and the second is used for data normalization of the signal in each well of the microplate. Using uterine myocytes, we assessed oxytocin (OT)-stimulated MLC(20) phosphorylation measured by ICW and WB, both using NIR fluorescence. ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT.Conclusion/significanceICW and WB yield comparable biological data. The advantages of ICW over WB are its high-throughput capacity, improved precision, and reduced sample preparation requirements. ICW might provide better sensitivity and precision with low-quantity samples or for protocols requiring large numbers of samples. These features make the ICW technique an excellent tool for the study of phosphorylation endpoints. However, the drawbacks of ICW include the need for a cell culture format and the lack of utility where protein purification, concentration or stoichiometric analyses are required.

CC BYText from Europe PMC. Highlighted sentences are the source of results.

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In this study, Oxytocin was a lab tool or a carrier, part of a mix, or the subject of a lab method. These results are not evidence about taking Oxytocin.

01LabNot a test of Oxytocin aloneIn vitro (cells)Other
In primary uterine myocytes, in-cell western and Western blot gave comparable signal linearity, specificity and time course of oxytocin-stimulated MLC20 phosphorylation.
In plain wordsIn lab tests on uterine cells, two measuring methods gave comparable results for oxytocin-triggered protein phosphorylation.
Peptide
Oxytocin
Outcome
Not applicableAgreement of in-cell western and Western blot for oxytocin-stimulated MLC20 phosphorylation
Study
n: not reported in source
Primary uterine myocyte cultures
Outcome group
Receptor activity and mechanism · Reproductive and sexual health
Source sentences
Summary
ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT.
1 context sentence(design, population, supporting numbers)
Summary · context
Using uterine myocytes, we assessed oxytocin (OT)-stimulated MLC(20) phosphorylation measured by ICW and WB, both using NIR fluorescence.
In this study, Oxytocin was a lab tool or a carrier, part of a mix, or the subject of a lab method. This result is not evidence about taking Oxytocin.
Show in the study text Report a problemFND-2037635801

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Version 2Current
11 Oct 2026, 01:30 UTC
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Version 1
9 Oct 2026, 02:53 UTC
First added · source update from Europe PMC

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