I am working on characterization of Mannose-1-Phosphate Guanylyltransferase enzyme (M-1-P GT) from Mycobacterium tuberculosis, by colorimetric method. For this i uses the following reagents in reaction,
Tris-Hcl (pH 7.5)
Mannose-1phosphate 2mM (Substrate of enzyme)
GTP 10mM (Substrate of enzyme)
MgCl2 4mM
pyrophophatase (PPase)0.004U/ul
M-1-P GT (enzyme)
H2O (final vol 50 ul)
As in the presence of enzyme (M-1-P GT) the above mentioned substrates converted into products (GDP-mannose and PPi). These PPi in the presence of PPase converted into 2Pi which can be detected by malachite green dye (malachite green is yellow color dye which turned green upon reaction with Pi) and reading could be taken by spectrometer.
I run the Test (which includes all of the above reagents) and Control (which includes all of the above reagent except M-1-P GT enzyme). so theoretically in the absence of enzyme no substrates could convert into product and malachite green couldn't turned into green color. therefor control should have less reading on spectrometer as compare to control. but i am experiencing the intensive color change in even control (without enzyme) which is false reading.
so kindly suggest me what could be the possible reason of this false reading in control?
and what should i do to solve this problem?
Thank you
My suggestions would be step by step. First of all : if you do not add the sample and you experience change in color this may be normal. But normal if it is "small" the change and stopping after some time. Does color change stop after a while? Otherwise may be something in the atmosphere (oxygen?) is affecting the change of color.
Also I would suggest to make tests by omitting always the sample and then do mixtures omitting in each test one of the components to see which one would be affecting color in the control.
Hope it helps
Hi there,
Are you sure about the quality of the phosphate containing reagents? If free phosphate is present because hydrolysis of these, it would lead to high OD readings...
The problem is almost certainly the high background from the enormous GTP concentration. Even a small percentage of phosphate in the GTP will result in a high background. The Malachite Green reagent is also very acidic, which causes the GTP to break down, releasing more phosphate. Malachite Green/molybdate reagents, in my experience, are sensitive to phosphate in the single- to double-digit micromolar range. Keep the GTP concentration to no more than 300 µM (100 µM would be better). Be prepared to try different sources of GTP to find one with a low background of phosphate.
If you really need to use mM GTP concentrations, you need a less sensitive phosphate assay. Try the MESG/purine nucleoside phosphorylase method. Another useful aspect of this method is that it gives a continuous readout, so you can get the whole progress curve from one reaction.
Thanks to all for your valuable suggestion especially Mr.Rafael Franco as your suggestion work for my experiment. I sorted out the reason of false reading that was my substrate Mannose-1-Phosphate. After optimizing the concentration of M-1-P i found the significant OD difference between my "Contro" & "Test", but still there is slight change in color of control. Does it OK for journal publisher to accept these results
A woudl need some more details to gave a 100% sure answer but for what you tell I am quite confident that you found the right way and that the journal will publish your results if including positive and negative controls.
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