Nupage mes sds buffer

Samples derived from 10 μg of Ft protein (~1 × 10⁸ cells) were mixed with Laemmli sample buffer and boiled for 10 min prior to resolution through 4–12% gradient SDS-PAGE pre-cast gels (Invitrogen). The running buffer was NuPAGE MES SDS buffer from Invitrogen; gels were variously run at 90–160 V. Resolved gels were stained with either Coomassie blue (BioRad) or transferred to nitrocellulose membranes. Coomassie-stained gels were scanned into Adobe Photoshop using an HP 2820 scanner. Membranes were blocked for 30 min with PBS, 0.05% Tween 20, 5% non-fat dry milk. Primary Abs were applied for overnight incubation at dilutions ranging from 1:1,000 to 1:60,000. Blots to be probed multiple times were first probed with mAb prior to stripping and reprobing with polyclonal sera. HRP-conjugated secondary Abs were used at dilutions ranging from 1:1,000 to 1:20,000. Occasionally the secondary conjugate was a biotinylated goat α-species-specific Ab followed by a tertiary conjugate of streptavidin-linked HRP. Development of the chemiluminescent substrate (SuperSignal West Pico, Pierce, Rockford, IL) was visualized using an Alpha Innotech imaging system in movie mode. Densitometric analysis of developed blots was performed on the same system.

Whole cell samples containing 10 μg of Ft protein (~1x10⁸ cells) were mixed with Laemmli sample buffer and boiled for 10 min prior to resolution through 4–12% gradient SDS-PAGE pre-cast gels (NuPAGE Bis-Tris, Invitrogen). The running buffer was NuPAGE MES SDS buffer from Invitrogen; gels were run at 120 V. Resolved gels were stained with either Coomassie blue (BioRad) or transferred to nitrocellulose membranes. Coomassie-stained gels were scanned into Adobe Photoshop using an HP LaserJet Pro 300 color (MFP-m375nw) scanner. Membranes were cut into sections, each containing one lane of WT and one lane of wbtA. Membrane sections were blocked in parallel for 30 min with PBS, 0.05% Tween 20, 5% non-fat dry milk. Rabbit sera (α-PBS, α-S4ΔguaBA prime, α- S4ΔaroD prime, α- S4ΔguaBA prime-boost, α- S4ΔaroD prime-boost) were applied in parallel for overnight incubation at a dilution of 1:1000 in PBS, 0.05% Tween 20. HRP-conjugated goat anti-rabbit secondary Ab (Southern Biotech 4010–05) was used at a dilution of 1:5000 in PBS, 0.05% Tween 20. Development of the chemiluminescent substrate (SuperSignal West Pico, Pierce, Rockford, IL) from the 5 individual membrane sections was visualized simultaneously using an BioRad ChemiDoc Touch Imaging System.

Samples derived from 10 µg (~1 × 10⁸ cells) of Ft were mixed with Laemmli sample buffer (BioRad, Hercules, CA, USA) and boiled for 10 min prior to resolution through 4–12% gradient SDS-PAGE precast gels (Invitrogen, Carlsbad, CA, USA). The running buffer was NuPAGE MES SDS buffer (Invitrogen). Gels were run at 90–160 V. Resolved gels were stained with either Coomassie blue (Bio-Rad) or transferred to nitrocellulose membranes. Coomassie-stained gels were scanned into Adobe Photoshop. Membranes were then blocked for 30 min with PBS, 0.05% Tween 20, 5% non-fat dry milk. Primary Abs were applied for overnight incubation at dilutions ranging from 1:500 to 1:60,000 in PBS-Tween. Following five washes with PBS-Tween, the membranes were incubated for 1 h with agitation with biotinylated goat anti-mouse IgG (H + L, Southern Biotech, Birmingham, AL, USA). Following an additional five washes with PBS-Tween, the membranes were incubated for 1 h with agitation with streptavidin-conjugated horseradish peroxidase (Southern Biotech). Development of the chemiluminescent substrate (SuperSignal West Pico, Pierce, Rockford, IL, USA) was visualized using an Alpha Innotech imaging system in movie mode. Densitometric analysis of developed blots was performed as previously described (7 (link), 9 (link)).