In this work, we developed a BN-PAGE protocol for the analysis of membrane protein complexes of C. thermocellum. Results and Discussion Preparation of Membrane Protein Samples Purification of protein complexes in an intact form (i.e. complete
with all peripherally associated proteins) is largely dependent on the solubilization conditions used and can differ for various complexes. By testing four commonly used detergents at different concentrations (see “”Methods”"), we were able to select a protocol using the detergent n-dodecyl-D-maltoside (DDM). This protocol detected a number of complexes in the molecular mass range from 60 to over 1,000 kDa. The molecular mass of protein complexes was calculated by plotting the MWs of marker LY3023414 cell line proteins against their migration distances. To identify the individual proteins in each complex, see more the one-dimensional BN gel strips were analyzed in the second dimension by SDS-PAGE, Figure 1. Putative complexes were consequently resolved into vertical “”channels”" enabling visualization of the individual constituents. this website Proteins that had formed a complex in the BN gel were tentatively recognized by their locations on a vertical line on the SDS gel, and also by their similar shapes on the SDS gel (as a
result of co-migration in the BN gel). Figure 1 Coomassie blue-stained 2D BN/SDS-PAGE separation of membrane protein complexes of C. thermocellum. Approximately 40 μg of protein was loaded in the first dimensional BN-PAGE lane. Sizes of molecular mass markers are indicated on the top of BN-P|AGE gel and at the left of the SDS gel. The slice of first dimensional BN-PAGE separation gel was placed on top of the second dimensional SDS-PAGE gel and resolved. Protein spots picked for mass spectrometry analysis are marked by arrows and numbered. Protein Identification Thirty six spots were picked from the SDS gel for MALDI-TOF/TOF identification. Thirty proteins were identified in 28 spots (Figure 1), and they represent 24 different proteins (Table 1). Among
them, 9 proteins were predicted by TMHMM [11, 12] (transmembrane hidden Markov model, http://www.cbs.dtu.dk/services/TMHMM/) to be fantofarone membrane protein containing α-helical transmembrane segments. The rest maybe membrane-associated proteins (described below). Many atypical membrane proteins are tethered to the membranes through lipid moieties, hydrophobic patches, charge interactions or by their association with a membrane protein complexes. The identified proteins were organized into functional groups based on COG using COGnitor tool available at NCBI [13, 14] and transporter related proteins were organized in membrane transporter complexes. Putative protein complexes and their estimated sizes observed on the BN-PAGE were summarized in Table 2. The false positive rate of protein identification was calculated by reverse database search to be lower than 2.5%. Table 1 Putative membrane proteins of C.