Dot Blot Method
1. Dot 1 µl of the desired sample; wait about 30 sec, then dot an additional 1ul over the
first.
Note: if you are testing several different samples using the same primary and
secondary antibodies, you can dot onto a single piece of nitrocellulose that has
been divided into a grid. You can do the incubations in an appropriate sized Petri
dish. If you are testing different concentrations of primary and secondary
antibodies with the same sample, dot your sample onto separate pieces of
nitrocellulose that are cut so that each piece fits into a 24 well plate.
2. After the final dot has been applied, allow the nitrocellulose to dry completely.
3. Block the nitrocellulose with agitation for 1 hour at room temperature.
4. Prepare the primary antibody in the block that was used. Leave the primary antibody
on the nitrocellulose overnight at 4oC with agitation.
5. Wash the nitrocellulose 8X, 5 minutes each with PBST or TBST (Depending on what
the block was made in).
6. Prepare the secondary antibody (HRP-conjugate) block. Leave the secondary
antibody on the nitrocellulose for 1.5 hours at room temperature with agitation.
7. Wash the nitrocellulose 8X, 5 minutes each with PBST or TBST
8. Rinse the nitrocellulose 5X PBS
9. Detect results with ECL as for Westerns.