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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.