I want to quantify the activity of an unknown specimen-derived protease mix via western blot with a single standard protein. ELISA does not work because partially digested fragments will still bind the antibody. What should I use as a loading control? Every spike-in protein would be degraded by the mix I assume.
Hi Johannes, have you tried zymography? It can be semi-quantitative depending on which class of proteinases you are looking at.
it is a good and interesting method, but I am not sure if I could use it in my case. I have a unknown mix of lot of unknown proteases. so I can't assume that some bands are not digested and could be used to normalize the proteolytic effect.
I was wondering if there are commercially available proteins and respective antibody available that would be resistant to all kinds proteases. Or something artificial, (dye or so) that could be used as a spike-in and would be transferred onto the membrane quantitatively and could be visualized on the membrane.
I think it would be a good start to identify the proteinases. Gelatin zymography has gelatin incorporated into the SDS PAGE gel. Gelatin is cleaved by most proteinases and after incubation and staining, the proteinases leave a clear band on the gel on a blue background.
Now you know the MW of the proteinases. Next is to determine their class.
You basically have 4 classes of proteinases. 1. Serine proteinases 2. Cysteine proteinases 3. Metalloproteinases and 4. Aspartic acid proteinases.
The most prominent are serine- and metalloproteinases. Many serine proteinases are covalently inhibited by PMSF. Add that inhibitor to a sample and you knock out most common serine proteinases. EDTA is a reversible inhibitor of metalloproteinases. Iodoacetamide is an irreversible inhibitor of cysteine proteinases.
You don't have to worry about Asp-proteinases because they usually have very low activity at neutral pH.
Thank you for your answer. I actually already know that we have all four protease types in the mix. It has been published for this specimen. My question is not to find out which proteases are in there but to determine the overall proteolytic activity with my target protein. So I don't want to use something like azocasein that can be used in a photometric assay but use a western blot to follow the degradation of my target dependent on time or different protease mix concentrations in the assay. For that I need a loading control to normalize the bands' intensities. At the moment I am thinking of using something as a spike-in control that has reasonable size, no protease sensitive peptide bonds and an antibody commercially available. Like an antibiotic for example but maybe bigger in size.
OK, I am a bit confused.
Is our target protein is a proteinase and you want an assay that specifically measures its activity?
Or is your target protein cleaved by proteinases and you want to measure its cleavage with time?
In either case, there is no natural protein that I know of that is not cleaved by proteinases (unless there is a protein out there that is made of only D-amino acids).
Yes the target is cleaved by the proteases and I want to measure its amounts. Ideal would be something that is not a protein, bigger than 5 kDa and there are antibodies. I saw that some use cyclosporin, unfortunately has inhibitory properties towards protease.
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