Chemidoc touch images

Proteins were quantified using the DC Protein assay Kit (Biorad) and separated on SDS-PAGE (stacking 5%, resolving 12% acrylamide). For Western blotting, proteins were separated on SDS-PAGE minigel (stacking 5%, resolving 12% acrylamide) and transferred on nitrocellulose membrane (0.45 µm) using trans-blot turbo blotting system (Biorad). Membranes were blocked with TBS containing milk (5%), then blotted with primary antibodies αMsp A (rabbit), αATP synthase beta (chicken) from Abcam, αHyt 27 (mouse) diluted in TBS containing BSA (5%). After washes with TBS containing Tween 20 (0.02%), secondary antibodies (αrabbit-HRP (goat) from Santa Cruz Biotech, αchicken-HRP (goat) from Abcam and α-mouse-HRP (goat) from Biorad) were incubated for 1 h. After washes, chemiluminescence was detected using the Chemidoc Touch Images (Biorad).

Lipoglycans (LAM, LM and PIM) were analyzed by SDS-PAGE (stacking 5% and resolving 15% acrylamide). Samples, containing the same amount of proteins, were digested by trypsin (Promega) at 37 °C overnight before the addition of the loading buffer. Gels were stained with silver staining^{43 (link),44 (link)}. Gels were scanned with Chemidoc Touch Images (Biorad) and analysed with Image Lab.