I want to perform a study to observe the effect of stress condition on enzyme activity of bacterial strain both Gram positive and Gram negative strain. In addition how can I measure total protein content in bacterial cell. Also I want to run SDS- PAGE for protein. So please give me detail about its methodology as well as some important literature. Thanking you.
Here is a great paper out of Mamadou Daffe's lab where they test the activities of several enzymes from the cell envelope of Mycobacteria.
http://www.ncbi.nlm.nih.gov/pubmed/9493394
I don't remember if they tested for the specific enzymes you are interested in, but it could give you an idea of how to approach your question.
Dear Umakant,
for these stress enzymes, you can lyse the treated cells, either by sonication or by enzymatic methods in protein extraction buffer supplemented with proteiase inhibitors, centrifuge the homogenate at 4 C , collect the supernatant for enzymatic assays. The supernatant will contain all the cellular as well as extracellular enzymes.
quantify the total protein using BSA as standard by Bradford or other methods, load the 50 ug of protein to SDS gel. Surely you will get the results.
Thank you vikas sir. Can you suggest me how can i induce oxidative stress in bacterial culture.
- Badges
- Science method