Agarose isoelectric focusing can improve resolution of membrane proteins in the two-dimensional electrophoresis of bacterial proteins.
2-D separation of bacterial membrane proteins is still difficult despite using high-resolution IPG-IEF/SDS-PAGE. We were searching for alternative methods to avoid typical problems such as precipitation, low solubility, and aggregation of membrane proteins in the 1-D separation with IPG-IEF. Blue native electrophoresis (BNE) and agarose IEF (A-IEF) were tested for their separation capacity and their capability of replacing IPG-IEF in the first dimension. SDS-PAGE was chosen for the second dimension on account of its outstanding resolution. We could confirm that only A-IEF was a useful replacement for the IPG-IEF in the first dimension resulting in 2-D protein distributions with additional membrane protein spots not being found after IPG-IEF/SDS-PAGE. A second interesting result was that the agarose IEF mediates the possibility of separation of membrane proteins in a partially native state in the first dimension. This native A-IEF resulted in drastically changed spot patterns with an acidic shift of nearly all spots and divergent distribution of proteins compared to non-native A-IEF and IPG-IEF. We found out that native and non-native A-IEF are powerful tools to supplement IPG-IEF/SDS-PAGE.
Altenhofer
Pia
P
Medical Faculty, Institute of Physiological Chemistry, Martin Luther University, Halle, Saale, Germany. pia.altenhofer@medizin.uni-halle.de
Schierhorn
Angelika
A
Fricke
Beate
B
D001426:Bacterial Proteins / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000302:isolation & purification*; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D012685:Sepharose
D001426:Bacterial Proteins; D008565:Membrane Proteins; D012685:Sepharose