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LifeTein Dot Blot protocol | LifeTein Peptide Blog
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. Pro
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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)