Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis.
Sample preparation is a fundamental step in proteomic methodologies. The quality of the results from a proteomic experiment is dependent on the nature of the sample and the properties of the proteins. In this study, various pre-treatment methods were compared by proteomic analysis; we analysed various rat brain structures after chloroform/methanol, acetone, TCA/acetone and TCA protein precipitation procedures. The protein content of the supernatant was also examined by 2-DE. We found that for four of the rat brain structures, precipitation with chloroform/methanol and acetone delivered the highest protein recovery for top-down proteomic analysis; however, TCA precipitation resulted in good protein separation and the highest number of protein spots in 2-DE. Moreover, TCA precipitation also gave high efficiency of protein recovery if prior sonication procedure was performed.
Fic
Ewelina
E
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland. ewelina.fic@uj.edu.pl
Kedracka-Krok
Sylwia
S
Jankowska
Urszula
U
Pirog
Artur
A
Dziedzicka-Wasylewska
Marta
M
D000096:Acetone / Q000737:chemistry; D000818:Animals; D001923:Brain Chemistry; D005591:Chemical Fractionation / Q000379:methods*; D011232:Chemical Precipitation; D002725:Chloroform / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D007525:Isoelectric Focusing; D000432:Methanol / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D014238:Trichloroacetic Acid / Q000737:chemistry
D009419:Nerve Tissue Proteins; D000096:Acetone; D014238:Trichloroacetic Acid; D002725:Chloroform; D000432:Methanol