I'm currently characterizing an endolysin. To test its efficacy in various environments, I have been utilizing a dye release assay to assess catatlytic activity. My issue is I've found this to not work well with gram positive bacteria, since the dye is covalently bonding with the actual PG and causing interference with the endolysin itself. I'm looking for new protocols. It was suggested that I use an antibody against PG. Has anyone done this before? Do you have any tried and trusted methods?
Hi,
I analyze PG with the use of SLP test. It is quite sensitive, but not specific test (it can measure also beta-glucans). I use protocol proposed by producer.
I found also ELISA test for PG, but I haven't used it yet. I will try it in a future.
Regards. Marcin Cyprowski
My work is mainly focused on gram-positive peptidoglycans (PGN), especially PGN from S. aureus.
When I get you right, you want to check, if and where your endolysin is cutting the PGN, right?
I also used this SLP kit, but just to quantify the PGN amount. But I think, in your case, classical PGN digestion assays with a subsequent RP-HPLC analysis, like we do here, would be the best way to determine any enzyme activity or also to identifiy the minimal cleavage site your enzyme needs. First, you would need PGN crude extract (the whole PGN molecule) to digest it with your enzyme and to check the HPLC pattern, you get. You can determine the peaks you get by MS.
The SLP reagent works relatively well, but I question its sensitivity in certain situations. Alternatives include a NOD2 reporter assay (invivogen), dry weight from lyophilized PG using a very sensitive/analytical scale, or MurNAc quantification Article Mass spectrometric quantitation of muramic acid, a bacterial...
- Badges
- Science topic
- Similar topics
- Biological Science
- Zoology
- Ornithology