I am checking several protocols and all of them use leupeptine in their buffers, and I don´t understand how is possible to accurately assess the calpain activity if you are inhibiting it...thanks
Marcelino: Lysis buffers contain protease inhibitor cocktails because you want to preserve the intact and functional calpains. The zymogram would not work if calpains were degraded in the lysis buffer before you ran your gel. Since leupeptin is a peptide inhibitor, it will disaggregate from intact calpain during electrophoresis. Since casein is homogenous throughout the gel, the intact calpain enzymes are free to cleave it and the effect is visible at the migration site on the gel.
If you want to study the effect of calpain inhibition in a zymogram, then you can use a specific, non-peptiditic calpain inhibitor. For example, see the article below from Wang:
http://ac.els-cdn.com/S0003986185712842/1-s2.0-S0003986185712842-main.pdf?_tid=cd80fc4c-b9fe-11e5-a71b-00000aacb360&acdnat=1452694152_4450b3ca19adadb7cb59e4e09fca395b
Thank you very much for your help. I took a look to the article and I have understood the idea about the leupeptin, I think everything is based on the inhibitor binding´s nature, I mean, if it is reversible or tight binding.
Anyway, about your explanation I don´t understand the point you make about peptidic and non-peptidic inhibitors, I mean, what would be the difference? It´s probably a silly question but I am new on this, I appreciate any help.
Marcelino:
You are correct. What I meant was indeed whether the inhibitor was reversible or irreversible. A reversible calpain inhibitor would disassociate from the calpain binding site and migrate at a different rate in the gel, while an irreversible inhibitor would stay calpain bound and inhibit casein digestion.
Thank you Brian, I will definitively use it when I run my experiment, hope it works.
Leupeptin is peptide -aldehyde - it does work on calpains, but not with the highest affinity
this was the original paper:
Raser, K.J., Posner, A. and Wang, K.K.W. (1995) Casein zymography: a method to study µ-calpain, m-calpain and their inhibitory agents. Arch. Biochem. Biophys. 319, 211-216.
pls check out.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides