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

Collaborators and Supporters

Team:XJTLU-CHINA/Protocols Dot blot