Hello everyone,
I'm trying to perform Proteinase K assay for downstream analysis of the mitochondrial localization of a protein of interest. I isolated mitochondria from HeLa cells (using mitochondrial isolation kit) and performed Proteinase K assay by adding proteinase-K in increasing concentrations to the isolated mitochondria, with and without Triton-X. I performed western blot to detect the protein of interest and other marker proteins (OMM-Tom20, IMS-Tim23, IMM-cytc, Matrix-Hsp60). In the control (without proteinase K), I could detect all the protein bands. But once I add proteinase-K, I'm not able to detect any of the proteins (including matrix protein Hsp60).
What could be the possible reason for this? Is the integrity of mitochondria lost while isolation? If so, please provide suggestions to maintain the integrity while isolating mitochondria..
You need to stop the reaction using PMSF
see my paper that has this protocol
Article Smac Mimetic with TNF- Targets Pim-1 Isoforms and Reactive O...
Hi Goodwin G. Jinesh ,
Thank you for your answer, I did add 10 mM PMSF after 30 minutes incubation with Proteinase K. I used proteinase K at increasing concentrations from 100 μg/mL to 1mg/mL.
Hi, I think you are using too much PK. I had the same problem once. I did serial dilutions and chose the minimum concentration showing degradation of membrane protein but not matrix protein. If I remember correctly, the concentration was around 0.5 ug/ml, but you should check that, as the activity of the enzyme can change from batch to batch.
Hi David Pla-Martín,
Thank you so much for your answer!!
I'll try reducing the concentration of PK by serial dilutions and optimize it!
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