3.4. Western Blot for Detection of Caspase-1 Proteolysis

Using the total protein concentration obtained in Subheading 3.1, step 1, load between 3 and 15 μg of protein into a 4–12% gradient gel. Resolve the samples by running the gel in 1× MES running buffer at 175 V and transfer to a PVDF membrane as described above.

Block membrane for 1 h in Blocking solution and probe overnight for caspase-1 by shaking at 4 °C using anti caspase-1 rabbit polyclonal antibody [7] in Blocking solution in a 1:10,000 dilution.

Wash excess primary antibody from membrane with 1× TBS-T three times and probe with secondary polyclonal goat antirabbit IgG (H + R) + HRP in a 1:20,000 dilution. Wash excess secondary antibody with 1× TBS-T four times. Signal visualization can be obtained as in Subheading 3.3, step 6. Using the gradient gel and the antibody described in step 2 enables the visualization of both procaspase-1 and cleaved caspase-1 (see Note 16).

In this case, β-actin can also be used as a loading control. Follow the same steps as summarized on Subheading 3.3, step 7.