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Open Access Methodology article

Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane

Mansi R Khanna1, Bruce A Stanley2 and Graham H Thomas1*

Author Affiliations

1 Departments of Biology and of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802 USA

2 Section of Research Resources, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA

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BMC Genomics 2010, 11:302  doi:10.1186/1471-2164-11-302

Published: 12 May 2010

Additional files

Additional file 1:

Golgi and post-Golgi proteins in head microsomes fractionated by density gradient centrifugation followed by two phase affinity partitioning at 5.7% PEG/Dextran. A - Fractionated microsomes prepared from heads and probed for the Golgi protein Lava lamp. Two isoforms of Lava lamp are detected (arrows) at ~170 kDa and ~315 kDa. On average Golgi membrane is heavier than the peak ER fractions as expected [16]); double headed arrow; see Figure 3), but some overlap is seen especially with the larger isoform which has a bimodal distribution. B - Fractionated microsomes prepared from heads and probed with anti-Horseradish Peroxidase (HRP). The epitopes recognized by anti-HRP depend on the presence of N-glycan core α1,3-linked fucose [46] and thus detects proteins in trans-Golgi and post-Golgi compartments. The prominent epitope at 42 kDa is thought to be our PM marker Nervana [28]. Trans- and post-Golgi proteins detected by anti-HRP extend from the Golgi fractions through to the lightest region of the gradient as seen with the fully glycosylated Nervana isoforms (see Figure 3A). C - Fractionation by two phase affinity partitioning following an initial density gradient fractionation and probed with anti-HRP. The most prominent band behaves the same way as Nervana (see Figure 6B) and probably is Nervana (see [28]). An overexposure of the final ConA eluate (ConA over) is included to show that other anti-HRP detectable proteins are also present in the PM fraction. Labeling: same as figure 3 for A and B and figure 4 for C. Loading: Equivalent amounts of all fractions were loaded.

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Additional file 2:

MudPIT identification of proteins purified by combination of density gradient centrifugation and 2PAP from Drosophila head microsomes: This table includes a list of all the proteins purified by our optimized protocol and identified with > 95% confidence. The sub-cellular compartment in which each protein can be found is indicated, along with the number of peptides identified. For single-peptide identifications, the sequence, precursor m/z and score of the peptide have been provided. Cross-references to Additional Files 3 and 4 are also included.

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Additional file 3:

Spectra for proteins identified by single-peptide hit: This table provides the matched peptide and spectrum for all single-peptide identifications.

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Additional file 4:

Hydropathy plots for proteins predicted to have transmembrane domains: This table provides the hydropathy plots of all those proteins predicted to have transmembrane domains by the method of Kyte and Doolittle.

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Additional file 5:

Functional categorisation of proteins identified as residents of the plasma membrane: This table classifies the plasma membrane proteins listed in Additional File 2 on the basis of their cellular function.

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