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Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics
Molecular Biology / Dot Blot Protocols 1
Introduction: A Dot blot (or Slot blot) is a technique in molecular biology used to
detect biomolecules. It represents a simplification of the northern blot, Southern blot, or western blot methods. In a dot blot thebiomolecules to be detected are not first separated by chromatography. Instead, a mixture containing the molecule to be detected is
applied directly on a membrane as a dot. This is then followed by detection by either nucleotide probes (for a northern blot and Southern
blot) or antibodies (for a western blot).
The technique offers significant savings in time, as chromatography or gel electrophoresis,
and the complex blotting procedures for the gel are not required. However, it offers no information on the size of the target biomolecule.
Furthermore, if two molecules of different sizes are detected, they will still appear as a single dot. Dot blots therefore can only
confirm the presence or absence of a biomolecule or biomolecules which can be detected by the DNA probes or the antibody.
A radioactive
sample can be hybridized to it allowing the researcher to detect variation between samples. The DNA is quantified and equal amounts
are aliquoted into tubes in excess of the number of its targets in the samples, such as 10ug for a plasmid and 1ug for a PCR amplicon.
These are denatured (NaOH and 95°C) and added to the wells where a vacuum sucks the water (with NaOH and NH4OAc) from underneath the
membrane (nylon or nitrocellulose).
Dot Blot Protocol
Materials
PVDF membrane
Wash Buffer
Blocking agent
Primary antibody
Secondary antibody
Detection reagent (ECL)
Place 10-20µL of protein solution (diluted using sample loading buffer)
on the membrane.
Allow the protein solution to AIR DRY (be patient, wait till the protein spot gets completely dried).
Block the membrane
with specific blocking agent.
Wash the membrane 3X, 5 min each with wash buffer.
Primary antibody titre:
Make different dilutions
of the antibody starting from 1µg/mL.
Use the wash buffer to make the dilutions.
Incubate the membrane with a primary antibody dilution
for 1hr.
Wash the membrane 3X, 5 min each with wash buffer.
Incubate the membrane with corresponding secondary antibody, for 1hr.
Incubate membrane in appropriate amount of ECL detection reagent.
Drain the excess
detection reagent and seal the membrane in a seal-a-meal cover.
Expose the membrane to an X-ray film and develop the film.
Other
Protocols for Dot Blot (Next Page)
