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)