I am working on one of the Mtb proteins, PPE18 whether it inhibits inflammasome activation or not? For that, I am trying to develop a western blot for pro and mature forms IL-1 beta. I got a good band for pro-IL-1 beta but I didn't get any band for mature form and also facing the same problem with caspase-1?
Here I can briefly mention the conditions that I followed to run western blot for pro and cleaved forms of both IL-1 beta and caspase-1: 1.Took protein concentration as 40ug. 2. Run 12% SDS-PAGE gel at 100V for nearly 2hrs 3. Preactivated the 0.45um PVDF blot in methanol 4. Done overnight transfer at 100mA/30V 5. After transfer, check the efficiency of transfer in all lanes with Ponceau S.Got clear protein bands in each and every lane 6.Done blocking with 5% TBST-milk for one hour at RT and done all washes with TBST three times each for 5 min 6. Used fresh both primary(1:1000 in TBST milk,incubated over night) & secondary antibodies( 1:2000 in TBST milk,incubated for 1 hr at RT) . For the second time, add 20mM of DTT to the protein pellet that precipitated from the supernatant by using methanol and chloroform. Then I got a good result for both pro and cleaved forms of IL-1 beta but still, I am facing the problem with caspase-1( both in lysate and supernatant).