2-D differential membrane proteome analysis of scarce protein samples.
Proteome studies with small sample amounts are difficult to perform, especially when membrane proteins are the focus of interest. In our study a new method for the analysis of scarce membrane protein samples combining large gel 2-D-CTAB/SDS-PAGE with fluorescence dye saturation labelling (satDIGE) was developed, allowing a highly sensitive differential analysis of different cell states. After Triton X-114 phase partitioning, enriched membrane protein samples of T cells were labelled at cysteine residues using fluorescence dyes and separated by large gel 2D-CTAB/SDS-PAGE. For a differential analysis 3 mug protein was found to be sufficient to detect proteins in a widespread well-separated diagonal spot pattern.
Helling
Stefan
S
Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
Schmitt
Edgar
E
Joppich
Cornelia
C
Schulenborg
Thomas
T
Müllner
Stefan
S
Felske-Müller
Stephanie
S
Wiebringhaus
Thomas
T
Becker
Gabriele
G
Linsenmann
Gudrun
G
Sitek
Barbara
B
Lutter
Petra
P
Meyer
Helmut E
HE
Marcus
Katrin
K
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D020543:Proteome; D021241:Spectrometry, Mass, Electrospray Ionization
D005456:Fluorescent Dyes; D008565:Membrane Proteins; D020543:Proteome