FtsZ is a tubulin homolog in bacteria, important in cell division. I need to establish its GTPase activity and inhibition assay.
I would be greatly helped with a valuable protocol/paper/correspondence for researchers working on this domain.
Thanks in advance.
Hi Muhammad,
Here is one option:
http://www.lifetechnologies.com/order/catalog/product/E6646
Read it carefully and let me know if you have questions.
Best.
Hi Muhammad,
For this type of assays you have select the way of monitoring the GTP hydrolysis. You can either monitor production of GDP or Pi, or degradation of GTP, or both.
You can monitor production of Pi in real time using coupled reactions and commercial kits such as the one copy-pasted below. Alternatively, you can use radioactively labeled GTP either at alpha, beta, or gamma phosphates. There are options to detect the hydrolysis, but thin layer chromotography seems to be the most appropriate for most cases.
Good luck.
EnzChek® Phosphate Assay Kit
(Molecular Probes®)
Citations (75)
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Description
The EnzChek® Phosphate Assay Kit provides a fast and sensitive spectrophotometric method for the quantification of inorganic phosphate in solution and for phosphate released during ATPase or GTPase enzymatic reactions.
See our complete line of Fluorescence Microplate assays.
• Sensitivity of 2 to 150 µM phosphate
• Spectrophotometric microplate-based assay detects a change in absorbance maximum from 330 nm to 360 nm
• Continuous monitoring of phosphate generated by ATPase or GTPase activity
In the presence of inorganic phosphate, the 2-amino-6-mercapto-7-methylpurine riboside (MESG) substrate is converted by the purine nucleoside phosphorylase (PNP) enzyme to the ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine product. This enzymatic conversion of MESG results in a spectrophotometric shift in maximum absorbance from 330 nm for the substrate to 360 nm for the product.
Sensitivity of the assay is in the range of 2 to 150 µM inorganic phosphate (2 to 150 nanomoles phosphate in a 1 mL volume), and the reaction can be performed over a pH range of 6.5 to 8.5. The EnzChek® phosphate reaction is sufficiently fast and quantitative for use in stopped-flow kinetic experiments.
had your issue been solved?
If not i can suggest some , since i am also working on Mtb-FtsZ .
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