I'm currently trying to induce mature IL-1B (p17) from J774 macrophages using LPS and ATP or Nigericin.
After treatment with LPS for 4 hours, I add into the culture 5mM ATP or 5uM Nigericin for another 1/2 hour, as what most published papers detail.
I then perform a western blot on the lysate and supernatant, and I load 20ug protein per well. I'm sure most of the protein in the supernatant as quantified by Bradford Assay is due to the FBS. But I just use 20ug as a reference. I run the lysate and supernatant on a 15% gel, transfer using a wet transfer for 80V, 80mins. Primary Antibody overnight and secondary antibody 1hr room temp, with 3 washes in between.
After exposing the blot with ECL solution from millipore, I can only visualize the pro-IL1B (p31) but not the mature IL-1B (p17).
Would like to kindly ask if anyone has tried inducing and visualizing mature IL-1B from J774 macrophages?
I'm not sure why I am not able to do so. Am wondering if it's my treatment procedure, me not concentrating the supernatant, western blot transfer time, or something else. Any help will be greatly appreciated! Thanks in advance. Cheers!