Hello, I am a masters student and I am working on plant-growth-promoting rhizobacteria. One of the assays that I am conducting is the ACC-deaminase assay. I am following the method and recommendations of Penrose and Glick (2003) (Attached). However, when I try the standard curve, my absorbence results are very low (as compared with the paper) and are also not linear. I am unsure why this is the case.
The method entails the following: I make a stock solution of alpha-ketobutyrate (100mM) in 0.1M Tris-HCl (pH 8.5). This is then diluted to 10mM with the same buffer. From this I make a series of alpha-ketobutyrate solutions ranging from 0.1 to 1.0 micromolar. The reaction mixture contains the following: 80 uL of the standard solution and 120 uL of the 2,4-dinitrophenylhydrazine (DNPH) reagent (0.2% 2,4-DNPH in 2M HCl). This mixture is then incubated at 30oC for 30 minutes. Afterwards, I add 800 uL of 2M NaOH and mix the solution. I then transfer 200 uL to the well of a microtiter plate and then read the absorbance at 540nm using a microtiter plate reader. For a blank (zero) I substitute 80 uL of the Tris-HCl buffer for the alpha-ketobutyrate solution in the above reaction mixture.
My first problem is that the blank has an absorbance similar to that of the standard reaction solutions. Therefore after I substract the blank absorbance from the standards I get very low values (including some negative values). However, the paper states that alpha-ketobutyrate in the range of 0-1.0 micromolar absorbs in the range of 0-1.6 at 540nm. Secondly, there is no linearity in my values, for example, the 0.2 uM alpha-ketobutyrate will have a higher absorbance than the 1.0 uM solution.
I have tried this twice (using fresh solutions) but I am obtaining the same results.
I am very unsure of what I am doing wrong, so could anyone who has had experience with this assay please help me out? Please see attached file
Regards,
Kyle
Dear Kyle,
I am not familiar with the particular method, but it seems to me that the bank needs to include all the reagents used except the substance of intereset, e.g., alpha-ketobutyrate.
Good luck!
I'm not familiar with this assay, but I agree, sometimes microplates are more variable than reading it in a cuvette.
At some level, the results are similar, so if you need just a semi-quantitative screen, the microplate reader may be good enough for you. As is suggested above, you can convert from plate-read values based on a spectrophotometer standard curve.
In a different spectrophotometric assay that I tried, it was interesting to find that the EXACT same samples read on a spec (in disposable plastic cuvettes) gave perfectly linear standard curves, while when I transferred the samples to a 96 well plate, the standard curve was non-linear. It may be that I just need to buy higher-end microplates. This test was done on a brand new, expensive, microplate reader- so most likely I can blame the plates, not the reader. I didn't pursue troubleshooting it- I just did my assay on a spec and got my result.
This kind of direct comparison is something easy for you to try, though. That way you can see if the problem is with your assay or with your plates. Good luck.
I´m not familiar with this method. First, be sure not to add the standard to the blank. Second, revise the quantities you add as you changed the method described by Penrose and Glick; Third, you need standard curves when the answers are not linear as seems to be this case; Fourth, try with the original method of Honma and Shimomura (1978), sometimes authors do not give all the modifications they made.
Good luck!
i'm not familiar with the method you working with but i think you should try another spectro. if you are very sure that the method is correct or check your glassware and all equipment for possible contamination. i had something like this with mine.
Hi
Did you find the solution? I am a PhD student and have the same problem with acc deaminase assay..
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