A Dot Blot technique is a simple and quick immunoassay that may be employed to determine if your antibodies and detection system are effective. It involves the application of a small amount of sample directly onto a membrane, which is then probed with a specific antibody or nucleic acid probe to detect the target molecule. Dot Blot may also be used to determine appropriate starting concentration of primary antibody for Western blot.
Dot Blot Protocol
Materials and Reagents Required
- Nitrocellulose Membrane
- Recombinant Proteins
- Cell Lysates (If required: Western Blot Protocol for Cell Lysates)
- Primary Antibodies
- TTBS (Tris, NaCl, Tween-20)
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Dot Blot Assay Protocol
- Use a strip of nitrocellulose membrane.
- Blot (10 µl) of different concentrations of recombinant protein onto membrane.
- Blot (10 µl) of different concentrations of cell lysates onto the membrane.
- Blot 10 µl of 100 µg/ml of primary antibody onto membrane.
- Incubate the membrane for 1 hour at room temperature. Ensure that the blots are dry before going to the next step.
- 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.
- Incubate the membrane with primary antibody for 1 hour at room temperature in TTBS.
- Wash the membrane 3 times (10 minutes each) in TTBS on rocker.
- Incubate the membrane with secondary antibody for 1 hour at room temperature in TTBS.
- Wash the membrane 3 times (10 minutes each) in TTBS on rocker.
- Detect with Western Glo Chemiluminescent detection reagents.
- Expose to film.