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Dot Blot Protocol

Disclaimer

This procedure should be used as a guideline only. Please keep in mind that Creative Biolabs cannot guarantee specific results for dot blotting assays.

Dot blotting is a technique used to detect, analyze, and identify proteins, which is similar to western blotting, but differs in that protein samples are not separated by electrophoresis, but are spotted directly onto a membrane or paper substrate by means of a circular template. If you have a purified protein and a specific antibody, you can use the dot blotting method to make semi-quantitative estimates of protein concentrations in crude preparations such as culture supernatants. Creative Biolabs can develop specific antibodies for you to use to perform dot blotting assays.

Preparation

  1. TBS is made of 20 mm Tris-HCl and 150 mm NaCl with a pH of 7.5.
  2. TBS-T is a TBS solution containing 0.05% Tween 20.
  3. BSA/TBS-T is a TBS-T solution containing 0.1% BSA.
  4. Nitrocellulose membrane

Procedure

  1. Prepare the nitrocellulose membrane. Draw a grid with a pencil to indicate the area where you want to make the blot.
  2. Use a narrow-mouthed pipette tip and spot 2 µL of sample onto the nitrocellulose membrane at the center of the grid. Apply slowly, minimizing the area of solution penetration (usually 2-4 mm in diameter).
  3. Allow the membrane to dry.
  4. Block non-specific sites in 10 cm Petri dishes with 5% BSA in TBS-T by immersion (30 min to 1 h at room temperature).
  5. Incubate for 30 min at room temperature with primary antibody (purified antibody 0.1-10 µg/mL, antiserum 1:1,000-100,000, hybridoma supernatant 1:100-10,000) in BSA/TBS-T.
  6. Wash three times with TBS-T (3 x 5 min).
  7. Incubate for 30 min at room temperature with the secondary antibody conjugated to HRP. For optimal antibody dilution, follow the manufacturer's recommendations.
  8. Wash three times with TBS-T (1 for 15 min, 2 for 5 min) and then once with TBS (5 min).
  9. Incubate with ECL reagent for 1 min, cover with Saran wrap (to remove excess solution from the surface), and expose to X-ray film in a dark room. Try several exposures of different lengths.
  10. Compare the signal of your unknown sample with the signal of the standard sample and estimate its concentration.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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