Abstract
BackgroundThe ‘phosphate-binding tag’ (phos-tag) reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC) in cultured human uterine myocytes.Methodology/Principal FindingsWe have evaluated and validated the concept that, when using an antibody (Ab) against the total-protein, the sum of all phosphorylation states in a single lane represents a ‘closed system’ since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT) and calpeptin (Calp) induce RLC kinase (MLCK)- and rho-kinase (ROK)-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA) induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.Conclusion/SignificanceWe have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional ‘loading’ or ‘reference’ standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.
Highlights
The cellular responses mediated by protein phosphorylation are vast in number and function [1], and a variety of biochemical techniques has been developed to study this important cell signaling modality [2]
We demonstrated that phosphorylation of regulatory light chain (RLC) at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here
Several studies have shown that three distinct phospho-states of RLC are detectable in smooth muscle (SM) preparations [5,18,19,20,21,22,23,24,25]
Summary
The cellular responses mediated by protein phosphorylation are vast in number and function [1], and a variety of biochemical techniques has been developed to study this important cell signaling modality [2]. The power and utility of this technique has recently been strengthened by the development of a dinuclear metal complex ‘phosphate-binding tag’ (phos-tag) that can be incorporated into the polyacrylamide gel matrix prior to SDS-PAGE [3,4]. This modification of traditional SDS-PAGE, promotes a physical separation of phospho-proteins proportional to the phosphorylation stoichiometry. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC) in cultured human uterine myocytes
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
