Analytical and Bioanalytical Chemistry

, Volume 398, Issue 1, pp 547–554

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

  • Kathryn L. Gilroy
  • Sarah A. Cumming
  • Andrew R. Pitt
Original Paper

DOI: 10.1007/s00216-010-3908-0

Cite this article as:
Gilroy, K.L., Cumming, S.A. & Pitt, A.R. Anal Bioanal Chem (2010) 398: 547. doi:10.1007/s00216-010-3908-0

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.

https://static-content.springer.com/image/art%3A10.1007%2Fs00216-010-3908-0/MediaObjects/216_2010_3908_Figa_HTML.gif
Figure

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.

Keywords

Quantum dotProtein A/GMultiplexingWestern blot

Abbreviations

HRP

Horse radish peroxidase

ECL

Enhanced chemiluminescence

QDot

Quantum dot

QDx

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

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Kathryn L. Gilroy
    • 1
  • Sarah A. Cumming
    • 1
  • Andrew R. Pitt
    • 1
  1. 1.Integrative and Systems Biology, Faculty of Biomedical and Life SciencesUniversity of GlasgowGlasgowUK