LifeTein Dot Blot protocol #

Dot Blot protocol
Dot blot is similar to the Western blot technique. However, the proteins are spotted directly onto the membrane or paper for detecting and analyzing. This is a good technique to estimate protein concentration.

Procedure
1. Prepare the nitrocellulose (NC) membrane.
2. Spot 2 µl of diluted samples onto the NC membrane and let it dry.
3.Blocking: Block the membrane in 1% BSA or non-fat milk in TBST (1 hr, Room Temperature).
4. Primary Antibody: Incubate with the primary antibody in BSA/TBST for 1 hr at RT.
5. Washing: Wash three times with TBST (3 x 5 min).
6.Secondary antibody: Incubate with secondary antibody conjugated with HRP for 1 hr at RT.
7. Washing: Wash three times with TBST (15 min x 1, 5 min x 2), then once with TBS (5 min).
8. Developing: Incubate with ECL reagent for 1 min and expose X-ray film in the darkroom with different lengths of exposure.

Reagents
TBS:
20 mM Tris-HCl
150 mM NaCl
pH 7.5

TBST:
0.05% Tween20 in TBS
BSA/TBST
0.1% BSA in TBST
NC: Nitrocellulose membrane (BIO-RAD)