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Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling

Lookup NU author(s): Kate Wilson, Alexander Keith, Dr Christopher Morris

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Abstract

Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 μg total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 μg total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA.


Publication metadata

Author(s): Wilson KE, Marouga R, Prime JE, Pashby DP, Orange PR, Crosier S, Keith AB, Lathe R, Mullins J, Estibeiro P, Bergling H, Hawkins E, Morris CM

Publication type: Article

Publication status: Published

Journal: Proteomics

Year: 2005

Volume: 5

Issue: 15

Pages: 3851-3858

Print publication date: 01/10/2005

ISSN (print): 1615-9853

ISSN (electronic): 1615-9861

Publisher: Wiley

URL: http://dx.doi.org/10.1002/pmic.200401255

DOI: 10.1002/pmic.200401255

PubMed id: 16145713


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