Western blot analyses were performed using standard methods. Briefly, samples were supplemented (or eluted from agarose beads) with NuPAGE LDS Sample Buffer (ThermoFisher Scientific). Unless indicated otherwise samples were supplemented with 50 mM DTT (reducing conditions). NuPAGE® Novex® 4%−12% Bis-Tris Gels (ThermoFisher Scientific) were used for SDS-PAGE. PageRuler Plus Prestained Protein Ladder (ThermoFisher Scientific) was used as molecular weight marker. Separated proteins were transferred to PVDF membranes (EMD Millipore, Hayward, CA) and membranes were blocked with 5% non-fat dry milk in TBS/0.1 Tween 20 (TBST/milk). Membranes were incubated with primary antibodies diluted in TBST/milk or TBST/5% BSA (0.1 – 1 μg/ml). HRP-conjugated primary or secondary antibodies were diluted 1:10000 in TBST/milk. After antibody incubations (1h - overnight) blots were washed 3x with TBST for 10 min each. Bound antibodies were visualized using SuperSignal West Pico/Femto Chemoluminescent Substrates (ThermoFisher Scientific) and exposure of AccuRay Blue X-Ray Films (E&K Scientific).
Supersignal west pico femto chemoluminescent substrates
Manufactured by Thermo Fisher Scientific
SuperSignal West Pico/Femto Chemoluminescent Substrates are sensitive substrates used for the detection of horseradish peroxidase (HRP) in Western blotting applications. They generate a chemiluminescent signal that can be detected using a compatible imaging system.
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2 protocols using supersignal west pico femto chemoluminescent substrates
1
Western Blot Protein Analysis Protocol
2
Western Blot Protein Analysis Protocol
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Western blot analyses were performed using standard methods. Briefly, samples were supplemented (or eluted from agarose beads) with NuPAGE LDS Sample Buffer (ThermoFisher Scientific). Unless indicated otherwise samples were supplemented with 50 mM DTT (reducing conditions). NuPAGE® Novex® 4%−12% Bis-Tris Gels (ThermoFisher Scientific) were used for SDS-PAGE. PageRuler Plus Prestained Protein Ladder (ThermoFisher Scientific) was used as molecular weight marker. Separated proteins were transferred to PVDF membranes (EMD Millipore, Hayward, CA) and membranes were blocked with 5% non-fat dry milk in TBS/0.1 Tween 20 (TBST/milk). Membranes were incubated with primary antibodies diluted in TBST/milk or TBST/5% BSA (0.1 – 1 μg/ml). HRP-conjugated primary or secondary antibodies were diluted 1:10000 in TBST/milk. After antibody incubations (1h - overnight) blots were washed 3x with TBST for 10 min each. Bound antibodies were visualized using SuperSignal West Pico/Femto Chemoluminescent Substrates (ThermoFisher Scientific) and exposure of AccuRay Blue X-Ray Films (E&K Scientific).
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