Need a NO SALT physiological buffer
Posted 15 October 2010 - 05:26 AM
Means: Cecal contents of mice are removed and diluted in 5 mLs PBS, then spun at low speed to remove large food particles. Supernatant then removed and spun at higher speeds to pellet bacteria. Cell-free supernatant used to measure ATP via a simple luciferase assay.
It is important that I measure ATP levels in the cecum separately from ATP levels in the bacteria (the latter is done via a separate experiment involving cell lysis). The problem is, the luciferase enzyme loses function when introduced to the cecal supernatant. I think this is due to the salts in the PBS buffer. I would like to resuspend the cecal contents in a physiological buffer that contains no salts, so as to keep the bacterial cells from lysing. Could I make a sucrose buffer for this purpose? What would the molarity be?
Thanks - I realize that this question may be appropriate for a different forum, but I'm a microbiologist so I wanted to try this one first.
Posted 17 October 2010 - 04:49 AM
Posted 17 October 2010 - 09:46 AM
The best buffer to preserve luciferase activity seems a tricine buffer, however I don't think PBS is going to inhibit luciferase activity. Perhaps is something in the cecal content. Are you sure it is luciferase inhibition? It is not that there is few free ATP in your cecal liquid? What happen if you introduce fresh ATP in the assay liquid?
Tricine buffer, you say? Thanks for the suggestion. You may be right that PBS does not inhibit luciferase activity -- I still need to perform a control where pure ATP is suspended in PBS and examined for luciferase activity. However, I did perform "spiked" controls where serial dilutions of cecal contents were were added to the pure ATP control (suspended in PBS) and it completely inhibited luciferase activity.
Posted 17 October 2010 - 04:41 PM