Laemmli sample buffer 4

To capture IgG-BCRs, 20 million Ramos B cells were washed with PBS, to remove FCS from the cell culture medium, and lysed in 8 ml PBS + 1% Triton-X100 for 60 min at 37 °C. IgG-BCRs were captured from cell lysis supernatants using 20 µl CaptureSelect^TM FcXL Affinity Matrix (Thermo Fisher Scientific) and an overnight incubation at 4 °C. B cell secreted IgG were captured from 12 ml Ramos B cell supernatant (cultured at a density of two million cells/ml) by a 4 °C overnight incubation with 20 µl CaptureSelect^TM FcXL Affinity Matrix (Thermo Fisher Scientific). Laemmli sample buffer (4×) (Bio Rad) was added to the IgG/FcXL bead slurry, boiled for 5 min at 95 °C, and loaded on a 4 to 15% SDS gel (Bio Rad). Proteins were detected with Coomassie Brilliant Blue G-250 Dye (Thermo Fisher Scientific) or the SilverQuest^TM Silver Staining Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The size was determined using the PageRuler^TM Plus Prestained Protein Ladder (Thermo Fisher Scientific).

Stationary-phase bacterial cultures (5 mL) were normalized to an OD₆₀₀ of 2.0, pelleted, and resuspended in 200 μL of diethyl pyrocarbonate (DEPC)-treated endotoxin-free water (GBiosciences no. 786-109). To corroborate that each sample contained approximately the same CFU level, 20 μL of the 200 μL resuspension was used for serial dilutions and plated to enumerate the real number of bacteria in the solution. Next, 2 μL of 2% SDS was added to the remaining 180 μL of sample and the solution was boiled for 5 min. Five microliters of proteinase K (New English BioLabs no. P8107S) was added to the reaction, and the samples were incubated at 59°C overnight. Ice-cold Tris-saturated phenol (Sigma no. 4557) was added (1:1), and the samples were vortexed and incubated at 70°C for 15 min. After cooling, the solution was centrifuged and a two-phase solution was observed. The top layer was harvested. Samples were mixed with Laemmli sample buffer 4× (Bio-Rad), and 15-μL aliquots were separated by denaturing gel electrophoresis using 4 to 15% polyacrylamide gradient gels (Tris-glycine-SDS buffer; Bio-Rad) and stained with ProQ300Emerald lipopolysaccharide gel staining kit (Molecular Probes) following the manufacturer’s instruction. Pictures of gels were taken with ChemiDoc MP imaging system (BIORAD), and the images were analyzed by Image Lab 6.0 Software (BIORAD).