Dot blotting

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.

4. 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 for Western 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.

5. 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 in TTBS. Wash the membrane 3 times (10 minutes each) in TTBS on rocker. Incubate the membrane with secondary antibody for 1 hour at room temperature in TTBS. Wash the membrane 3 times (10 minutes each) in TTBS on rocker. Detect with Western Glo Chemiluminescent detection reagents. Expose to film.

6. 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.

7. 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