Solubilization of trichloroacetic acid (TCA) precipitated microbial proteins via naOH for two-dimensional electrophoresis.
In preparing intracellular microbial samples for one- or two-dimensional electrophoresis, trichloroacetic acid (TCA) precipitation is frequently used to remove interfering compounds. Solubilization of TCA precipitate typically requires the addition of a number of chaotropes or detergents, in a multistep process, that requires hours to carry out. In this study, a simple, rapid, one-step method to solubilize TCA precipitated proteins is presented. Precipitated proteins are pretreated with 0.2 M NaOH for less than 5 min, followed by addition of standard sample solubilization buffer (SSSB). When compared to solubilization with SSSB alone, NaOH pretreatment of TCA-precipitated intracellular protein from Aspergillus oryzae and Escherichia coli shows an approximate 5-fold increase in soluble protein. In addition, two-dimensional gel electrophoresis on resolubilized proteins shows an equivalent number of proteins in samples with and without NaOH pretreatment.
Nandakumar
M P
MP
Department of Chemical & Biochemical Engineering, University of Maryland, Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, Maryland 21250, USA.
Shen
Jie
J
Raman
Babu
B
Marten
Mark R
MR
D001236:Aspergillus oryzae / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D020543:Proteome; D012972:Sodium Hydroxide / Q000494:pharmacology; D013997:Time Factors; D014238:Trichloroacetic Acid / Q000737:chemistry* / Q000494:pharmacology*
D010455:Peptides; D011506:Proteins; D020543:Proteome; D012972:Sodium Hydroxide; D014238:Trichloroacetic Acid