Novex tricine sds sample buffer

Bacteria were cultured to A₆₀₀ ~0.5. One milliltre was harvested by centrifugation, washed with PBS, then repelleted and resuspended in a volume of 1× Novex Tricine SDS sample buffer (Invitrogen) normalized for cell density (50 µl per 1 ml A₆₀₀ 0.5). Samples were boiled for 15 min and either cooled on ice (no proteinase K) or incubated with proteinase K (NEB) at 55 °C for 1 h. Samples were re-boiled and electrophoresed using the tricine buffer system with Novex tricine 16%-acrylamide gels (Invitrogen). Spectra Multicolor Low Range Protein Ladder (Thermo) was included to indicate approximate molecular weights. Gels were fixed, washed, stained using Pro-Q Emerald 300 (Invitrogen), and imaged using UV transillumination (Biorad Chemidoc MP). Gels were subsequently stained with Coomassie Brilliant Blue for detection of total protein. Image lab software (Biorad) was used to quantify LOS or total protein intensity levels. Samples were normalized by dividing the LOS intensity level of each band region by the total protein level from Coomassie staining. Relative values were calculated by dividing each normalized LOS value by the total normalized LOS levels in WT.

Different Aβ preparations were re-suspended in 10 µL of 2X Novex Tricine SDS sample buffer (Invitrogen, Carlsbad, CA) and boiled in water for 3 min. For Western blots, samples were electrophoresed on a Novex 10–20% Tricine gel with 1X Novex Tricine SDS Running Buffer (Invitrogen, Carlsbad, CA). Proteins were transferred onto a 0.2 µm PVDF membrane and the membrane was briefly fixed with 0.2% glutaraldehyde at room temperature (RT) for 30 min, blocked at RT in 1X Tris Buffered Saline plus 0.05% Tween-20 (TBST) containing 5% non-fat dry milk for 1 hr, and incubated overnight at 4°C in primary antibody (B436; 1∶1000 in TBST/5% BSA/0.02% NaN₃). On the second day, the membrane was washed three times with TBST and then incubated in secondary horseradish peroxidase (HRP) linked anti-mouse antibody (GE Healthcare Life Sciences, 1∶5000 in blocking buffer). Blot was washed with TBST and applied with enhanced chemiluminescence (ECL) for signal development. For non-Aβ-detection samples, Tris-Glycine gel and buffer system were applied.