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.
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