Dot Blot

Overview

Dot Blot is a kind of Western Blot (without having to run a gel and doing a transfer). It is mostly used to determine the antibody titre, before performing the actual Western Blot. Dot Blot helps to determine the amount of primary or the secondary antibody that is needed to run a CLEAN Western Blot.

Materials

• PVDF membrane
• Wash Buffer
• Blocking agent
• Primary antibody
• Secondary antibody
• Detection reagent (ECL)

Procedure

1. Place 10-20μL of protein solution (diluted using sample loading buffer) on the membrane.
2. Allow the protein solution to AIR DRY (be patient, wait till the protein spot gets completely dried).
3. Block the membrane with specific blocking agent.
4. Wash the membrane 3X, 5 min each with wash buffer.
5. Primary antibody titre:
   1. Make different dilutions of the antibody starting from 1μg/mL.
   2. Use the wash buffer to make the dilutions.
   3. Incubate the membrane with a primary antibody dilution for 1hr.
6. Wash the membrane 3X, 5 min each with wash buffer.
7. Incubate the membrane with corresponding secondary antibody, for 1hr.
8. Wash the membrane 3X, 5 min each with was buffer.
9. Incubate membrane in appropriate amount of ECL detection reagent.
10. Drain the excess detection reagent and seal the membrane in a seal-a-meal cover.
11. Expose the membrane to an X-ray film and develop the film.

Notes

1. Washing buffer is the one that is used for regular Western Blotting. NV