dot+blot+protcol

Dot Blot Protocol

Abcam dot blot protocol http://www.abcam.com/ps/pdf/protocols/Dot%20blot%20protocol.pdf

Description A technique for detecting, analyzing, and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate. Concentration of proteins in crude preparations (such as culture supernatant) can be estimated semiquantitatively by using “Dot Blot” method if you have both purified protein and specific antibody against it.

Reagents TBS: 20 mM Tris-HCl 150 mM NaCl pH 7.5 TBS-T: 0.05% Tween20 in TBS BSA/TBS-T: 0.1% BSA in TBS-T Nitrocellulose membrane (BIO-RAD, Trans-Blot, etc.)

Procedure below). grid. Minimize the area that the solution penetrates (usually 3-4 mm diam.) by applying it slowly. chamber. antisera, 1:100 to 1:10000 for hybridoma supernatant) dissolved in 5% BSA/TBS-T for 1 hr at RT. recommendation) for 1 hr at RT. surface), and expose X-ray film in the dark room. Try several different lengths of exposure.
 * Have nitrocellulose membrane ready, draw grid by pencil to indicate the region you are going to blot (see
 * Using narrow-mouth pipet tip, spot 2 µl of samples onto the nitrocellulose membrane at the center of the
 * Let the membrane dry.
 * Block non-specific sites by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). Use 10cm Petri Dish for reaction
 * It is possible that even better blocking can be achieved if the membrane is blocked overnight at 4 C.
 * Incubate with primary antibody (0.1-10 µg/ml for purified antibody, 1:1000 to 1:100000 dilution for
 * Wash three times with TBS-T (3 x 5 min).
 * Incubate with secondary antibody 1:10,000 in 5% BSA in TBST (for optimum dilution, follow the manufacturer’s
 * Wash three times with TBS-T (15 min x 1, 5 min x 2), then once with TBS (5 min).
 * Dry membrane (can be dried between blotting filter paper)
 * If the secondary antibody was an HRP antibody, then incubate with an HRP reagent.
 * If an ECL reagent was used then incubate for 1 min, cover with Saran-wrap (remove excessive solution from the
 * Detect antibody. If an AF647 secondary antibody was used, then the antibody can be detected on the Typhoon.
 * May want to compare the signal from your unknown sample to that of standard and estimate the concentration.