High reproducibility of large-gel two-dimensional electrophoresis.
Two-dimensional gel electrophoresis (2-DE) facilitates the separation of thousands of proteins from highly complex protein mixtures and has become a central method in proteomics in recent years. In the present study, we examined the technical variability of large 2-DE gels with respect to sample preparation, electrophoresis procedure, data acquisition, and biological variation by analyzing a disease (Huntington's disease) and control state with a commercially available software package, PROTEOMWEAVER trade mark. Scatter plots and correlation coefficients were obtained to quantify both technical and biological variation. Even 2-DE gels run separately in both dimensions yielded correlation coefficients around 0.88 and deviations from the mean close to 20% for low-intensity spots. This indicates a high technical reproducibility of the 2-DE procedure developed in our laboratory. Variability within a biological condition was low and comparable to technical variation (at least 0.87). Two-dimensional (2-D) gels obtained from samples of different biological conditions (health vs. disease) achieved a variability similar to intracondition and technical variability. These findings highlight the importance of multiple gel and spot-by-spot comparisons to identify biological significant changes. Minor errors introduced by technical and biological variation allow a comparison of all gels within a study which facilitates the tackling of complex biological problems.
Challapalli
Kiran Kumar
KK
Institute for Theoretical Biology, Humboldt University, Berlin, Germany.
Zabel
Claus
C
Schuchhardt
Johannes
J
Kaindl
Angela M
AM
Klose
Joachim
J
Herzel
Hanspeter
H
D000818:Animals; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification*; D009687:Nuclear Proteins / Q000235:genetics; D020543:Proteome; D015203:Reproducibility of Results; D012984:Software
C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D020543:Proteome