I am running an assay for 5'nucleotidase activity based on measurement of ammonia through Berthelot's method. I used AMP as a substrate which is cleaved by 5 nucleotidase to form adenosine. Adenosine is then deaminated through action of adenosine deaminase and ammonia is released. My reaction mix contains AMP, adenosine deaminase and 5' nucleotidase in PBS at pH 7.5 and reaction is carried out at 30 deg C before adding Berthelot's reagent. There is no activity in my reaction mix. Can anyone suggest if something need to be changed such as pH or type of buffer?
Thanks
Dear Vasundhra Bahl,
I have read the paper contained in the following link and found some important steps to be taken:
1-Temperature should be 37 degrees and not 30 C.
2-The color reading should be between 15-40 minutes
3-A factor of 1.04 should be taken into account
4-The Blank: The recommended method allows for the ammonia originally present in the serum as well as that generated from sources other than AMP during the
incubation, since the serum is incubated under conditions identical with those of the test but omitting substrate. It is also necessary to take account of ammonia and adenosine present as a contaminant of AMP. Further allowance is made for generation of adenosine from AMP during incubation at 37°C. All of the above sources of ammonia, other than that due to specific hydrolysis of AMP by AcPase
of serum, were fully allowed for in the control employed in the definitive assay.
5-The pH should be 5.6 (the optimal pH of the enzyme).
Please recheck if the steps I counted are correct.
http://jcp.bmj.com/content/24/6/493.full.pdf
You can determine the activity using a method by Wood & Williams (see the following link) and compare the results from the two different methods.
http://www.clinchem.org/content/27/3/464.full.pdf
hoping this will be helpful,
Rafik
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays