Dot Blot/Antibody Titration Protocol

Proteins are adhered to a nitrocellulose membrane (via dot blot) or PVDF membrane (via gel
electrophoresis and transfer chamber) and then cut into strips that are used to screen
combinations of serial dilutions of primary to secondary antibodies. For comparable results,
it is important to keep all protocol parameters the same, such as sample type, incubation
times, wash times and temperatures.

1. Prepare the protein sample in the desired buffer. The protein sample should contain
an abundance of the antigen of interest. Usually, the antigen concentration of a
sample is unknown, so a wide range of dilutions may need to be tested.
2. Cut a nitrocellulose membrane into 1cm strips, enough to test the number of
primary and secondary antibody dilutions to be screened. Label the strips of
antibody dilutions with pencil.
o If a PVDF membrane is used, then run the protein sample through gel
electrophoresis and transfer to the PVDF membrane. Do not let the PVDF
membrane dry out. Proceed to step 4.
3. Dot the protein dilutions onto the dry nitrocellulose membrane strips using as little
volume as possible for each dot (1-5 µl) so that sample does not spread across the
membrane. To dot samples greater than 5µl, dot smaller volumes multiple times on
the same spot allowing each dot to absorb and dry for 2-5 minutes. After dotting is
complete, allow the strips to completely dry for 10-15 minutes.
4. Block the membrane by incubating in blocking buffer for 1-2 hours at room
temperature. Use a shaker to ensure even coverage of non-specific sites on the
membranes.
5. Incubate the membrane strips with the appropriate primary antibody dilution in
washing buffer containing 10% volume of blocking buffer for 1 hour on an orbital
shaker. The strips that are receiving the same concentration of primary antibody
may be incubated together in the same bath.
o The typical starting primary antibody dilution is 1:1000, so test a series of
1:250, 1:500, 1:1000, 1:2000 and 1:4000 (0.2 to 5.0 µg/ml).
 If there is no recommended dilution then start with 1 µg/ml of
purified antibody.
 Negative control samples with no primary or secondary antibodies
should also be included to reveal non-specific cross-reactivity.

6. Wash the membrane strips thoroughly in wash buffer, 4 washes of 5 minutes.

7. Incubate the membrane strips with the appropriate secondary antibody dilution in
washing buffer containing 10% volume of blocking buffer for 1 hour on an orbital
shaker. The strips that are receiving the same concentration of secondary antibody
may be incubated together in the same bath.
o The typical starting secondary antibody dilution is 1:10,000, so test a series of
1:2,500, 1:5,000, 1:10,000, 1:20,000, and 1:40,000.
8. Wash the membrane strips again, as in step 6
9. Prepare the substrate working solution as described in the corresponding data
sheet.
10. Incubate the membrane strips with the substrate working solution for 5 minutes.
11. Protect the membrane strips by placing them in a plastic wrap.
12. Expose the wrapped strips protein side up against film. Exposure time can vary and
will likely take longer for lower concentrations of primary/secondary antibody or if
the membrane has aged past 24 hours. The resulting film of an optimized blot will
clearly yield a band for your protein of interest with no background and no non-
specific bands.
13. If problems persist and a chemiluminescent substrate was used, then it is possible to
strip and reprobe the membranes with adjusted dilutions of primary and secondary
antibodies. Keep in mind that stripping does remove some sample protein from the
membrane, especially nitrocellulose.