To assay the GST activity from liver lysate, I have used 0.5 mM GSH, 0.5 mM CDNB and 10 µl of tissue supernatant. I have assayed with the increase in the absorbance at 340 nm. I have set blank with 0.1 M potassium phosphate buffer with 1 mM EDTA . When I have added only GSH and CDNB (without tissue supernatant) there was an increase in the absorbance, which indicates the formation of GSH-CDNB adduct automatically. So, should I deduct the rate of GSH-CDNB adduct formation from the rate of enzymatically formed GSH-CDNB adduct? What should be the calculation to get the actual GST activity?
Not sure why are you getting increasing absorbance. Does the absorbance keep increasing over time? Is there any possibility that there is some residual tissue contamination in the assay?
I have checked for 5 minutes without tissue lysate, but still it showing an increase in the absorbance over time. Is there any possibility to form GSH-CDNB adduct without GST?
Dear Deba
The best way to determine whether your exp is working or not is to use pure GST at diff. conc. and derive a standard plot. how sure you are that the lysates you hav prepared have copious amount of functional GST. It is always recommended to start any enzyme estimations by using pure proteins.
Please refer this for calculations. http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/cs0410bul.pdf
and, yes you will get an increase in the blank reading
Subtract the (OD340)/minute of the blank from the
(OD340)/minute of the sample. Use this rate for the
calculation of the GST specific activity
Hope this info is helpful
Best
S
Dear Subhankar Das
I dont have any pure GST protein. I have assayed the GST activity on the basis of GSH-CDNB formation rate. I have two sets of tissue, i.e. control and treated one. How I will deduct the nonenzymatic formation of GSH-CDNB adduct from the enzymatic reaction rate? Can you kindly explain me? I am a bit confused with the calculation.
CDNB is similar in molecular composition to DNTB (aka Elman's reagent). When doing a GSH assay with DNTB as the colorometric indicator, my initial absorbance varied greatly between experiments. I subsequently determined that this was due to light exposure decomposition of DNTB. I suggest making CDNB fresh and protecting from ambient light exposure (use opaque test tube or wrap tube in aluminum foil). Maybe this will help you as well.
Best of luck,
Tyler
Dear Dr. Olkowski,
I have found an increase in the absorbance in the range of 0.038 to 0.039 per minute without tissue lysate.
I would consider this kind of increment as large and not acceptable. In my view this such change in absorbance is not be associated with your assay, but rather instrument drift. Such drift may be caused by aging light source (in your case UV lamp) or some unspecified electronic noise. What kind of spectrophotometer are you using?
Dear Dr. Olkowski,
I am using Cary 60 UV-Vis spectrophotometer, Agilent Technologies.
Dear Demarest,
I have performed the experiment by keeping CDNB in a amber tube. But, still I am getting an increase in absorbance.
As a first step of troubleshooting I suggest you run diagnostics procedure on your spec.
Could be the spec as Olkowski indicated. It could also just be nonspecific GS-CDNB reaction since GSH can degrade at temperatures higher than 4 degrees. You will need to subtract the blank as background regardless of how low you can get the blank/nonspecific reaction. What is the percentage of your bank delta abs/min compared to your samples and positive controls?
Dear Dr. Olkowski,
In case of blank, delta abs/min is 0.038 to 0.039. However, with sample the delta abs/min is around 0.0756. I used to perform the kinetic study at 25 °C. Before the experiment, I used to keep all the reagents in ice bucket.
One possibility you need to consider is that light path components may be dirty due to accumulation of lipid and protein residues. This type of contamination occurs frequently when tissue homogenates are used in assays. I strongly suggest you should run diagnostics to exclude the potential of electronic or light source problems. If all diagnostic parameters are OK, then perform optic path clean up and decontamination.
Dear Dr. Olkowski,
Definitely I will check all the contamination and spectro related parameters. Thank you very much for your kind suggestions.
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