A simple method to remove contaminating salt from IPG strips prior to IEF.
In this study, Saccharomyces cerevisiae yeast lysate is used to demonstrate how a simple wash procedure can improve IEF of IPG strips passively rehydrated in the presence of NaCl. By performing three 10 min washes after IPG strip rehydration and before IEF, corresponding second-dimensional gels from strips containing NaCl look similar to control strips while the second-dimensional gels of unwashed strips contains streaks and spaces devoid of protein. Up to 500 mM NaCl was added to the yeast lysate and successfully focused following this wash regime. Protein loss due to the washes was determined to be minimal by comparing replicates of washed and unwashed strips and analyzing the densities of their corresponding second-dimensional gel spots. In the event of unknown salt contamination, indicated by low voltage during focusing, it is possible to stop focusing, wash the strips, and then continue focusing with acceptable second dimension results.
Heppelmann
Carrie J
CJ
Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, MN 55902, USA.
Benson
Linda M
LM
Bergen
H Robert
HR
D002021:Buffers; D005591:Chemical Fractionation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000302:isolation & purification*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000737:chemistry / Q000302:isolation & purification
D002021:Buffers; D029701:Saccharomyces cerevisiae Proteins; D012965:Sodium Chloride