western blot protocols, troubleshooting, scientific resources and research atricles for the research community and science students
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Protein
detection using the dot blot protocol is similar to western blotting in
that both methods allow for the identification and analysis of proteins
of interest. Dot blot methodology differs from traditional western blot
techniques by not separating protein samples using electrophoresis. Sample
proteins are instead spotted onto membranes and hybridized with an antibody
probe. Semi-quantitative measurements can be made of the spots.
1. PubMed:
comprehensive database - 15 million citations
2. NIH:the National Institutes of Health
3. UniProt: The world's most comprehensive catalogue of information on proteins
4. Western
Blot Procedure Western blot protocol overview.
Available from
Amazon.com:
Dot
Blot Immunoassay Identifies True Lyme Infection. (Statistical Data Included)
: An article from: Family Practice News
b . Pipette 2µl from each fraction onto the membrane, allow the membrane.
c . When dry, incubate the membrane in blocking solution for 1 hour.
d . After incubation, incubate (rotate or shake) the membrane with primary antibody solution (diluted in blocking solution), for 2 hours at room temperature.
e . Wash the membrane in washing buffer (3 x 10 min).
f . Incubate the membrane with secondary antibody-alkaline phosphatase enzyme conjugate solution (in blocking solution) for 1 hour.
g . Wash the membrane in washing buffer (3 x 10 min).
h . Incubate the membrane in substrate solution, until spots are visible.
i . Stop the reaction – rinse the membrane in distilled water.
j . Air dry the membrane.