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Sample Preparation in Biological Mass Spectrometry pp 217–242Cite as

Two-Dimensional Polyacrylamide Gel Electrophoresis

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Abstract

Two-dimensional polyacrylamide gel electrophoresis (2-DE), when combined with mass spectrometry (MS) has the power to identify and quantify differential protein expression of large sample sets analysed in parallel. By separating proteins by isoelectric point (pI) and molecular weight (MW), it has the potential to detect 2,000–3,000 different protein spots from a single sample separation. With the development of technologies such as Difference In-Gel Electrophoresis (DIGE), which also allow the parallel addition of an internal standard within each gel, 2-DE has shown improved reproducibility and moreover improved protein detection sensitivity. Furthermore, 2-DE provides protein abundance information as well as pI and MW and so has the potential to resolve and visualise post-translational modifications such as phosphorylation, a major advantage over non gel-based separation techniques. In this chapter we review sample preparation procedures from prevention of proteolysis, including the potential of a novel protein stabilising device, to lysing of a given sample with an appropriate lysis buffer. We describe additional steps which may be taken in order to achieve a protein sample which will yield a highly reproducible 2-DE map with distinct protein spots. This chapter also discusses the basic methodology behind 2-DE and the various protein detection reagents which are available. Finally, we discuss how the 2-DE images can be quantified and differentially expressed proteins can be identified by MS.

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Abbreviations

2-DE:

Two dimensional electrophoresis

ACN:

Acetonitrile

ANOVA:

Analysis of variance

DIGE:

Difference gel electrophoresis

DTT:

Dithiothreitol

ECL:

Enhanced chemiluminesence

ELISA:

Enzyme linked immunosorbant assay

FDR:

False discovery rates

IEF:

Isoelectric focusing

IgG:

Immunoglobulin

IPG:

Immobilised pH gradients

LC:

Liquid chromatography

LCM:

Lasercapture microdissection

MALDI:

Matrix assisted laser desorption/ionisation

MW:

Molecular weight

PAGE:

Polyacrylamide gel electrophoresis

PCA:

Principle component analysis

pI :

Isoelectric point

PMSF:

Phenylmethylsulfonyl fluoride

PTM:

Post translational modification

SDS:

Sodium dodecyl sulphate

TCA:

Tricholoracetic acid

TOF:

Time of flight

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Authors and Affiliations

  1. School of Medicine and Medical Science, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland

    Aisling A. Robinson, Ciara A. McManus & Michael J. Dunn

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  1. Aisling A. Robinson
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  2. Ciara A. McManus
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  3. Michael J. Dunn
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Correspondence to Aisling A. Robinson .

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  1. , Department of Genetics and Complex Disea, Harvard School of Public Health, Harvard, Huntington Ave., SPH-1 665, Boston, 02115, Massachusetts, USA

    Alexander R. Ivanov

  2. , Research and Development, Pressure BioSciences, Inc., Norfolk Ave. 14, South Easton, 02375, Massachusetts, USA

    Alexander V. Lazarev

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Robinson, A.A., McManus, C.A., Dunn, M.J. (2011). Two-Dimensional Polyacrylamide Gel Electrophoresis. In: Ivanov, A., Lazarev, A. (eds) Sample Preparation in Biological Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0828-0_13

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