DOT - Dot Blot

Overview

Dot Blot (DOT) is a simple method that can be used for identifying proteins and determining starting concentrations for western blot, as well as analysis via templates directly onto a membrane or paper substrate.

Required materials

• Nitrocellulose membrane

• 5% dry milk

• TTBS (50 mM Tris, 0.5 M NaCl, 0.05% Tween-20, pH 7.4)

• Western Chemiluminescent detection

Protocol

1. Have nitrocellulose membrane ready, indicate the region you plan to use with a pencil.

2. Blot 5-10 μl 3 times of recombinant protein at different concentrations onto the membrane.

3. Blot 5-10 μl 3 times of Cell lysate at different concentrations onto the membrane.

4. Blot 10 μl of 100ug/ml of primary antibody onto the membrane.

5. Incubate for 1 hour 30 minutes, and ensure the nitrocellulose membrane is dry.

6. Use 5% dry milk in TTBS to block the membrane for 1 hour.

7. Drain off the blocking solution and immediately incubate the membrane in primary antibody TTBS at room temperature for 1 hour and 30 minutes.

8. Drain TTBS and agitate membrane in fresh TTBS for 10 minutes, repeat 3 times.

9. After the 3rd drain incubate the membrane for 1 hour 30 minutes in secondary antibody TTBS.

10. Drain TTBS and agitate membrane in fresh TTBS for 10 minutes, repeat 3 times.

11. Use Western Chemiluminescent detection reagent.

12. Expose to film.

Results

The analysis of the dot blot can be observed with the naked eye by looking for spots that have appeared indicating the presence of a protein. The agitate membranes can be analyzed digitally for precise measurements of concentrations and data collection.