Western blots

• Put the nitrocellulose membrane in 5 % milk blocking solution and gently shake at RT for 1 hr.

Blocking Solution (100 ml): 

• Dilute the antibody to the concentration recommended by the product usage information with 5 % milk blocking solution (around 2 ml)

• Suck off the blocking solution and add the diluted primary antibody to the membrane and shake it overnight at 4 ℃ 

• Suck off the primary antibody solution and then wash the membrane with washing buffer (Tris buffer salt solution (TBS) containing 0.1 % Tween-20) for 3 times at 15 min interval 

• Dilute the HRP-conjugated secondary antibodies in washing buffer and add it into the membrane and gently shake for 1 hr at RT

• Suck off the HRP-conjugated secondary antibodies solution and wash the membrane with washing buffer for 3 times at 15 min interval

• Incubate in ECL solution (3 ml Clarity Western Peroxide Reagent + 3 ml Clarity Western Luminol/Enhancer Reagent, freshly prepared, BIO-RAD 170-5061) for 5 min and wrap the dry membrane onto PE plastic film for developing

• Exposure time is within 2 minutes using X -ray film