a. Label nitrocellulose membrane (using a pencil) to identify protein elution fractions.

b . Pipette 2μl from each fraction onto the membrane, allow the membrane.

c . When dry, incubate the membrane in blocking solution for 1 hour.

d . After incubation, incubate (rotate or shake) the membrane with primary antibody solution (diluted in blocking solution), for 2 hours at room temperature.

e . Wash the membrane in washing buffer (3 x 10 min).

f . Incubate the membrane with secondary antibody-alkaline phosphatase enzyme conjugate solution (in blocking solution) for 1 hour.

g . Wash the membrane in washing buffer (3 x 10 min).

h . Incubate the membrane in substrate solution, until spots are visible.

i . Stop the reaction � rinse the membrane in distilled water.

j . Air dry the membrane.