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Milk blocking buffer compatibility with TMB and ECL substrate detection
Serial dilutions of HeLa cell lysate (7.5, 3.45, 1.88, 0.94, 0.47, 0.23 and 0.12μg) were prepared and separated by electrophoresis. The proteins were transferred to nitrocellulose membranes (Part No. 88018) and the membranes were blocked for 45 minutes with 5% skim milk in TBS + 0.05% Tween™ 20, Clear Milk Blocking Buffer 1X in ultrapure water (Part No. 37587) or StartingBlock T20 (PBS) Blocking Buffer (Part No. 37539). After blocking, the membranes were incubated with suggested concentrations of primary antibodies to targets A, B and C. The membranes were washed and then incubated with of HRP-Conjugated Goat Anti-Mouse IgG (Part No. 31430) and HRP-Conjugated Goat Anti-Rabbit IgG (Part No. 31460) (both at 0.067μg/mL) and then washed again. Working solution of Pierce ECL Substrate (Part No. 32106) was prepared according to the instructions and added to once set of membranes for 5 minutes. The membranes were removed from the substrate, placed in plastic sheet protectors and exposed to CL-XPosure Film (Part No. 34090) for 5 seconds. The other set of membranes were placed in 10mL of Pierce 1-Step Ultra TMB-Blotting Solution (Part No. 37574) and the color development was stopped at 5 minutes by rinsing the membranes with water.
Antibodies:
A. Heat Shock Protein 86 (Hsp86) (Part No. PA3-013)
B. Actin (Part No. MA1-744)
C. Cyclophilin B (Part No. PA1-027A)
