实验概要

Peprotech provides a development (immunostaining) protocol.

实验步骤

1. After the transfer is complete, incubate the membrane in blocking solution (3% Nonfat Dry Milk in diH₂O) for 30 minutes with gentle agitation on an orbital shaker.

2. Wash the membrane three times with TBST (TBS, pH 7.2 with 0.1% TWEEN-20) in a clean tray on an orbital shaker; each wash lasting 5-10 minutes.

3. Dilute the probing (primary) antibody in TBST to a volume of 50ml (approximate final concentration of 0.20µg/ml) and incubate the membrane in the temperature. (The optimum incubation time depends on the antibody/antigen binding affinity and must be pre-determined for each antibody.)

4. Wash the membrane three times as in step #2.

5. Dilute the secondary antibody in TBST according to the manufacturer's specification. Incubate the membrane in a clean tray containing 50ml of diluted secondary antibody for one hour at room temperature on an orbital shaker.

6. Wash the membrane three times as in step #2.

7. Color development requires the use of a commercially available (e.g. Bio-Rad or Sigma) alkaline phosphatase conjugate substrate kit. Follow the manufacturer's instructions.

8. After the bands become clearly visible, stop the color by placing the membrane in a tray containing diH₂O for at least ten minutes.