Abstract
Identification of the 329 spots visible in 2D gels of plant mitochondrial proteins is a challenge. This paper describes a 2D mini-gel protocol involving free-radical scavengers and purified reagents to make it compatible with protein sequencing, and evaluates its performance. The paper also describes a “FastBlot” sequencing cycle with the cycle time for protein sequencing from PVDF membranes reduced to less than 29 min with femtomole sensitivity. Other benefits of the cycle include reduced lag, reduced background, reduced loss of labile residues, and increased initial and repetitive yields. The procedure gave excellent results with maize mitochondrial proteins: of six protein spots that we tried to sequence, only one was blocked. The other spots yielded considerable sequence information. One spot was identified from the sequence as superoxide dismutase, while another spot corresponded to an unidentified cDNA from rice. The results of these experiments show that modifications of our previous procedures can provide good N-terminal protein sequencing from individual spots on 2D gels. The technique makes it possible to obtain sequence data, prepare gene probes, and identify many of the polypeptides in the 2D-gel map for plant mitochondria.
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Abbreviations
- 2D:
-
two-dimensional
- CAPS:
-
3-[cyclohexylamino]-1-propanesulphonic acid)
- ddH2O:
-
distilled-deionized water
- DPU:
-
N,N-diphenylthiourea
- IEF:
-
isoelectric focusing
- PVDF:
-
polyvinylidene difluoride
- TEMED:
-
N,N,N′,N′-tetramethyleneethylenediamine
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Dunbar, B., Elthon, T.E., Osterman, J.C. et al. Identification of plant mitochondrial proteins: A procedure linking two-dimensional gel electrophoresis to protein sequencing from PVDF membranes using a FastBlot cycle. Plant Mol Biol Rep 15, 46–61 (1997). https://doi.org/10.1007/BF02772112
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DOI: https://doi.org/10.1007/BF02772112