A simple, sensitive and selective quantum-dot-based western blot method for the simultaneous detection of multiple targets from cell lysates (original) (raw)

Abstract

Quantum dots (Qdots) are fluorescent nanoparticles that have great potential as detection agents in biological applications. Their optical properties, including photostability and narrow, symmetrical emission bands with large Stokes shifts, and the potential for multiplexing of many different colours, give them significant advantages over traditionally used fluorescent dyes. Here, we report the straightforward generation of stable, covalent quantum dot–protein A/G bioconjugates that will be able to bind to almost any IgG antibody, and therefore can be used in many applications. An additional advantage is that the requirement for a secondary antibody is removed, simplifying experimental design. To demonstrate their use, we show their application in multiplexed western blotting. The sensitivity of Qdot conjugates is found to be superior to fluorescent dyes, and comparable to, or potentially better than, enhanced chemiluminescence. We show a true biological validation using a four-colour multiplexed western blot against a complex cell lysate background, and have significantly improved previously reported non-specific binding of the Qdots to cellular proteins.

Stable covalent conjugates of Qdots with a range of emission frequencies and protein A/G have been generated. These can be bound to appropriate primary antibodies from many species and used for the selective detection of target proteins within a sample. We have demonstrated this using four-colour detection in a western blot format from a cell lysate.

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Abbreviations

HRP:

Horse radish peroxidase

ECL:

Enhanced chemiluminescence

QDot:

Quantum dot

QD x :

Quantum dot emitting at wavelength x

MEK:

Mitogen-activated protein kinase kinase

RKIP:

Raf kinase inhibitor protein

GST:

Glutathione-_S_-transferase

V5:

Simian virus 5 epitope tag—GKPIPNPLLGLDST

EDC:

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

NHS:

_N_-hydroxysuccinamide

PMT:

Photomultiplier tube

HA:

Hemagglutinin epitope tag—YPYDVPDYA

FRAP:

FKBP–rapamycin-associated protein

FKBP:

FK506 binding protein

FRB:

FKBP12–rapamycin-binding domain of FRAP

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Acknowledgements

We are grateful to Dr. Susan Gannon for help in the preparation of protein samples used in these experiments and Professor Walter Kolch for gifts of plasmids. This work was supported by the RASOR Interdisciplinary Research Collaboration, funded by BBSRC and EPSRC (BBC5115721), and the Scottish Funding Council.

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Authors and Affiliations

  1. Integrative and Systems Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, University Avenue, Glasgow, G12 8QQ, UK
    Kathryn L. Gilroy, Sarah A. Cumming & Andrew R. Pitt

Authors

  1. Kathryn L. Gilroy
  2. Sarah A. Cumming
  3. Andrew R. Pitt

Corresponding author

Correspondence toAndrew R. Pitt.

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Gilroy, K.L., Cumming, S.A. & Pitt, A.R. A simple, sensitive and selective quantum-dot-based western blot method for the simultaneous detection of multiple targets from cell lysates.Anal Bioanal Chem 398, 547–554 (2010). https://doi.org/10.1007/s00216-010-3908-0

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