Dot Blot Protocol

A Dot Blot is a simple and quick assay that may be employed to determine if your
antibodies and detection system are effective. Dot Blot may also be used to determine
appropriate starting concentration of primary antibody for Western blot.
1.
2.
3.
4.

Use a strip of nitrocellulose membrane.

Blot (10 l) of different concentrations of recombinant protein onto membrane.
Blot (10 l) of different concentrations of cell lysates onto the membrane.
Blot 10 l of 100 g/ml of primary antibody onto membrane.

5. Incubate the membrane for 1 hour at room temperature. Ensure that the blots are dry
before going to the next step.
6. Block the membrane with 5% dry milk in TTBS (50 mM Tris, 0.5 M NaCl, 0.05%
Tween-20, pH 7.4) for 1 hour at room temperature. Pour off the block buffer, but
keep membrane wet at all times for the remainder of the procedure.
7. Incubate the membrane with primary antibody for 1hr at RT in TTBS.
8. Wash the membrane 3 times (10 minutes each) in TTBS on rocker.
9. Incubate the membrane with secondary antibody for 1 hour at room temperature in
TTBS.
10. Wash the membrane 3 times (10 minutes each) in TTBS on rocker.
11. Detect with Western Glo Chemiluminescent detection reagents.
12. Expose to film.