Search Thermo Fisher Scientific
Milk blocking buffer compatibility with nitrocellulose and PVDF membranes
Serial dilutions of A549 cell lysate (5, 2.5, 1.25, 0.62, 0.31, 0.16 and 0.78μg) were prepared and separated by electrophoresis. The proteins were transferred to nitrocellulose (Part No. 88014) and PVDF membranes (Part No. 88585) and the membranes were blocked for 30 minutes with 5% skim milk in PBS + 0.05% Tween™ 20 or Clear Milk Blocking Buffer (Part No. 37587) prepared at 1X in ultrapure water. After blocking, the membranes were incubated with primary antibodies to targets A, B and C. The membranes were washed and then incubated with 10ng/mL of HRP-Conjugated Goat Anti-Mouse IgG (Part No. 31430) and 6.7ng/mL of HRP-Conjugated Goat Anti-Rabbit IgG (Part No. 31460) then washed again. Working solution of SuperSignal West Dura Extended Duration Substrate (Part No. 34076) was prepared according to the instructions and added to the 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.
Antibodies:
A. Heat Shock Protein 86 (Hsp86) (Part No. PA3-013) at 1μg/mL
B. NFκB (Millipore No. 06-418; 1mg/mL) at 1μg/mL
C. PP2A (Millipore No. 05-421) at 0.2μg/mL
