Analysis of the membrane integral proteome is mainly dependent on the ability of protein separation. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique capable of efficient membrane protein separation, so far mainly applied to the mitochondrial oxidative phosphorylation machinery. Applying BN-PAGE to the thylakoid membranes after mild solubilization with digitonin we succeeded in displaying the response of the green algae Chlamydomonas reinhardtii to altered culture conditions. In addition, by peptide mass fingerprinting and matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) extremely hydrophobic subunits of the photosystem complexes with 5-11 transmembrane helices were identified, which could not be accessed by in-gel digestion in previous studies.