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Dot Blot Protocol

The document describes the dot blot protocol for protein detection and analysis. Unlike western blotting, dot blotting does not separate protein samples using electrophoresis but instead spots protein samples directly onto membranes. The membrane is then incubated with primary and secondary antibodies to detect and make semi-quantitative measurements of protein spots, similar to western blot analysis. The document provides the step-by-step procedure for performing dot blot analysis.

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Surajit Bhattacharjee
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198 views1 page

Dot Blot Protocol

The document describes the dot blot protocol for protein detection and analysis. Unlike western blotting, dot blotting does not separate protein samples using electrophoresis but instead spots protein samples directly onto membranes. The membrane is then incubated with primary and secondary antibodies to detect and make semi-quantitative measurements of protein spots, similar to western blot analysis. The document provides the step-by-step procedure for performing dot blot analysis.

Uploaded by

Surajit Bhattacharjee
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOC, PDF, TXT or read online on Scribd
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doc 15 March 2007

Dot Blot Protocol Web www.westernblotting.org 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. Procedure a. !abel nitrocellulose membrane "using a pencil# to identify protein elution fractions. b . Pipette $%l from each fraction onto the membrane& allow the membrane to dry. c . When dry& incubate the membrane in bloc'ing solution for ( hour. d . )fter incubation& incubate "rotate or sha'e# the membrane with primary antibody solution "diluted in bloc'ing solution#& for $ hours at room temperature. e . Wash the membrane in washing buffer "* + (, min#. f . -ncubate the membrane with secondary antibody-al'aline phosphatase enzyme con.ugate solution "in bloc'ing solution# for ( hour. g . Wash the membrane in washing buffer "* + (, min#. h . -ncubate the membrane in substrate solution& until spots are visible. i . Stop the reaction / rinse the membrane in distilled water. . . )ir dry the membrane.

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