Have anyone tried IL-1alpha antibody for western blotting using mouse tissue samples? I have used abcam Il-1 alpha. As per the information sheet two bands were reported (one at 17 kDa and other at 23 kDa). However i found only one band at 17 kDa. What could be the possible reason?
All the IL-1's are cleaved upon activation by cysteine proteases. Either you don't have enough protease inhibitor in your protein lysis solution
OR those mouse brain tissues are going crazy since as soon as the mouse dies you'll get massive necrosis and tissue destruction, largely due to IL-1 and caspase cascades. If it's this one it's going to be hard to find full IL1a.
Do you perfuse your mice with fixative+protease inhibitors during sacrifice?
@cole .Thanks Cole for your feedaback. I haven't perfused mice with fixative. I dissected brain regions and immediately transferred to liquid nitrogen. Later I processed by homogenizing the tissue samples with Urea Buffer (8M). Further centrifuzed for supernatant and loaded into gel for PAGE. Gel run fine and even transfer (followed wet transfer method) was fine. Even I got Perfect Bactin bands. May be your point of protease inhibitor making all difference. I should try other lysis buffer rather than 8M urea buffer. Can you please share the working recipe for lysis buffer and any reference ? . I will be thankful.
I haven't tried this on harvested tissues but I use the Complete Protease inhibitor in RIPA buffer (Boston BioProducts, 1pill dissolved in 10ml RIPA). I also have a protease inhibitor that I add, but if you're just looking for full length caspase 9 you shouldn't need it. I've only used this lysis solution of cell cultures, NOT TISSUE, it might not be enough to lyse through connective tissues/high lipids/ etc.
I really think that the main problem youre facing is that right after the mice dies almost all the CNS tissues will start to die via caspase cascade. Thats why I think perfusion might help. Ill get back to you with a protocol for brain tissue sample prep. (Rabkin lab)
So you don't need to perfuse unless you really don't want the blood in your samples. After you sacrifice your mice just harvest the brains and mince them in a RIPA buffer supplemented with protease inhibitors.
Side note, are you sure your antibody is for full and cleaved IL1? You are running your samples in a SDS based loading dye AFTER boiling for 5min? In other words do you have a positive control for full length IL1, to make sure its not an antibody problem?
If this doesn't work I'd reach out to the company the Ab comes from and authors from papers that looked at IL1 in brain tissues( if any)
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