Team:XJTLU-CHINA/Protocols Dot blot
Dot Blot
Materials:
- TBS Buffer: 20 mM Tris-HCl; 150 mM NaCl; pH 7.5.
- TBS-T Buffer: 0.05% Tween20 in TBS.
- BSA/TBS-T Buffer: 0.1% BSA in TBS-T.
Procedure:
- Have nitrocellulose membrane ready, draw grid by pencil to indicate the region which are going to blot.
- Using a narrow-mouth pipette tip, spot 2 µL of sample onto the nitrocellulose membrane at the center of the grid. Minimize the area that the solution penetrates (usually 2–4 mm diameter) by applying it slowly.
- Let the membrane dry.
- Block non-specific sites by soaking in 5% BSA in TBS-T in a 10 cm Petri dish (30 min to 1 h at room temperature.
- Incubate with primary antibody (0.1–10 µg/mL for purified antibody, 1:1,000–1:100,000 for antisera, 1:100–1:10,000 for hybridoma supernatant) in BSA/TBS-T for 30 min at room temperature.
- Wash three times with TBS-T (3 x 5 min).
- Incubate with secondary antibody conjugated with HRP for 30 min at room temperature. For optimum antibody dilution, follow the manufacturer's recommendation.
- Wash three times with TBS-T (1 x 15 min and 2 x 5 min), then once with TBS (5 min).
- Incubate with ECL reagent for 1 min, cover with Saran wrap (remove excessive solution from the surface), and expose X-ray film in the dark room. Try several different lengths of exposure.
- Compare the signal from your unknown sample to that of the standard and estimate the concentration.
Applications in our project: Transformation of BBa_K2309027, BBa_K2309022 and BBa_K2309028
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