Dot Blotting, steps involved, blotting membrane , uses

1. DOT BLOTTING
- M.Meenakshi,
Assistant Professor,
Department of Microbiology,
Sri Ramakrishna College of Arts And Science for Women,
Coimbatore

2. DOT BLOTTING

3. METHODOLOGY
A general dot blot protocol involves spotting 1–2 microliters of a samples onto
a nitrocellulose or Polyvinylidene difluoride (PVDF) membrane and letting it air
dry. Samples can be in the form of tissue culture supernatants, blood serum, cell
extracts, or other preparations.
The membrane is incubated in blocking buffer to prevent non-specific binding. It
is then incubated with a primary antibody followed by a detection antibody or a
primary antibody conjugated to a detection molecule ( alkaline phosphatase).
 After antibody binding, the membrane is incubated with
a chemiluminescent substrate and imaged.

5. DOT BLOT PROTOCOL
A Dot Blot is a simple and quick assay that may be employed to determine if your
antibodies and detection system are effective.
 Dot Blot may also be used to determine appropriate starting concentration of
primary antibody forWestern blot.
Use a strip of nitrocellulose membrane.
Blot (10 µl) of different concentrations of recombinant protein onto membrane.
Blot (10 µl) of different concentrations of cell lysates onto the membrane.
Blot 10 µl of 100 µg/ml of primary antibody onto membrane.

6. DOT BLOT PROTOCOL
Incubate the membrane for 1 hour at room temperature. Ensure that the blots are dry
before going to the next step.
Block the membrane with 5% dry milk in TTBS (50 mM Tris, 0.5 M NaCl, 0.05% Tween-20,
pH 7.4) for 1 hour at room temperature. Pour off the block buffer, but keep membrane wet
at all times for the remainder of the procedure.
Incubate the membrane with primary antibody for 1hr at RT inTTBS.
Wash the membrane 3 times (10 minutes each) inTTBS on rocker.
Incubate the membrane with secondary antibody for 1 hour at room temperature inTTBS.
Wash the membrane 3 times (10 minutes each) inTTBS on rocker.
Detect withWestern Glo Chemiluminescent detection reagents.
Expose to film.

8. SUMMARY
Dot blot is conventionally performed on a piece of nitrocellulose membrane or
PVDF membrane.
After the protein samples are spotted onto the membrane, the membrane is
placed in a plastic container and sequentially incubated in blocking buffer,
antibody solutions, or rinsing buffer on shaker.
 Finally, for chemiluminescence imaging, the piece of membrane need to be
wrapped in a transparent plastic film filled with enzyme substrate.
Vacuum-assisted dot blot apparatus has been used to facilitate the rinsing and
incubating process by using vacuum to extract the solution from underneath the
membrane, which is assembled in between several layers of plates to ensure
good seal between sample wells, hold waste solution, and deliver suction force.
 For chemiluminescence signal detection, apparatus need to be disassembled
and the membrane need to be taken out and wrapped in a transparent plastic
film.

9. USES
Performing a dot blot is similar in
idea to performing a western blot,
with the advantage of faster speed
and lower cost.
Dot blots are also performed to
screen the binding capabilities of an
antibody