i am doing GSH assay by ellman method, for this i have prepared my standard curve from which i got equation (Y=0.766X-0.017, R2=.998). however we can also calculate the value using molar extinction coefficient value of GSH (in cuvette value is =13600). i want to know how i can calculate value for unknown sample. if i do with molar ext coff. and than what will the value for microplate since pathlenth is note 1 cm. moreover if i do with standard curve than how, please help me out i am getting confusion ?
Use the standard curve, prepared with the same volume as the unknown sample. The slope of the standard curve is the extinction coefficient for that pathlength. If the slope was 0.766 absorbance units per mM GSH, then the extinction coefficient was 0.766/mM (leaving off the pathlength because it is not known exactly and it is the same for all samples). For an unknow sample, use the equation of the standard curve to convert absorbance to concentration, where Y is absorbance: (Y+0.017)/0.766 = X mM.
Adam is right, from standard curve (in which ever units you expressed/calculated the amount of GSH measured - M, mol, mmol, ect.) just insert the obtained absorbance in formula that Adam wrote and you will obtain the amount of GSH per sample.
Hi Aadarsh,
I assumed a linear standard curve based on the information in the original question. The equation is just the equation of a line y=m*x + b where m, the slope, is the extinction coefficient; and b, the y-intercept, is the baseline.
Standard curves are not always linear and may require another equation.
this is a common problem for microplate based assays. You have identified the problem correctly. First determine path-length for the microplate. this is very easy to do. Calibrate your Elman assay using 1 cm colorimeter this gives 13600 (?). Repeat same measurements using microplate & SAME calibration solutions. Lets suppose microplate calibration slope is 6800. Thereafter the microplate path-length (L') = 6800/13600 = 0.5cm. The value for L' depends on assay volume so- keep same microplate assay volume for assays and calibration. Good luck. These principles are shown in the attaché paper: http://www.ncbi.nlm.nih.gov/pubmed/25529660
Some microplates (for example, the Epoch Biotek that we have in our lab) have the option "pathlenght correction", that in parallel with the reading of the sample perform some reading that the software use to convert directly each absorbance you had in each sample to its equivalent in a 1 cm cuvette. In the end, the software gives several readings for the same sample, including the real absorbance and the corrected absorbance. So, you can use the corrected absorbance directly in the calculation using the molar ext. coeff. So, see if your microplate reader has the same option. Hope it helps!
Dear JFS. Yes. Automated path length correction is available for high-end microplate readers. When not available proceed as explained in my last posting.
1. Sorry to miss another feature of the question from Soni. The calibration equation Y=0.766X-0.017, R2=.998 shows a slope of 0.766 because the x-axis data was in units of mM or uM (or similar). To get values of the calibration slope (uncorrected microplate extinction coefficient) please put your x-axis (GSH concentration) data in units of as mole/l; Using mM/ uM for plotting data is not fatal particularly for internal data handling but obviously - but not helpful where one wishes obtain "absolute" calibration values. 2. We assume that you wish to obtain the "real" extinction coefficient for the Elman's assay in order to compare with literature values.
Hi All,
I need your technical advice regarding my ongoing assays.
I need to use Megazyme pNP-oligosaccharides in couple of days. I have established pNP standard curve by taking pNP dilutions 0 to 1 mM (1 µmol/mL) in 20mM tris (pH ~6, optimum pH of my enzyme too). I added 1M sodium carbonate (Na2CO3) to all the samples in the standard range including blank. After mixing well, I took 300 ul of each in flat bottom clear 96 well plate, measured absorbance at 405 nm, 410 nm, and 420 nm on Spectramax plate reader. Yet, I have got best absorbance at 420 nm plotted against known concentrations at X axis with a good equation at 420 nm y = 0.9997x - 0.0321, R² = 0.9998. (so, 0.9997 may be an uncorrected extinction coefficient). From this equation, I can calculate unknown concentration of pNP released during assay. My standard curve equation is persistent by repeating with same plate and some plate and fresh solution. I believe that the equation should give me direct calculation of unknown concentration by putting absorbance (y) in the equation i.e. X (unknown Concentration mM) = Y (Absorbance at 420 nm) + 0.0321/ 0.9997
So, should I bother for exact extinction coefficient as people mention while working on normal spectrophotometer with 1cm length glass cuvette?
I assume extinction coefficient would be necessary I would do my assay by direct application of A = εlc
[where A is absorbance, ε is the molar extinction coefficient (which depends on the nature of the chemical and the wavelength of the light used), l is the length of the path light must travel in the solution in cm, and c is the concentration of a given solution]
What's your expert opinion?