Dear all,
I would be very grateful if one of you can detect the error of the following protocol to make the standard curve for measuring the activity of Pectinase, since the results obtained were wrong.
PROTOCOL:
First, I prepared a stock solution of 1mg/mL of D-galacturonic acid and prepared different concentrations 0.2, 0.4, 0.6 and 0.8 mg/mL
In tubes, I added 0.9 ml of 1% pectine dissolved in citrate buffer pH 5.0, then I added 0.1 mL of the different concentrations of D-galacturonic acid and heated the tubes at 40°C for 30min. Later, I added 1ml of DNS to all the tubes and boiled them at 100°C for 5min. After cooling, I measured in spectrophotometer at 540nm, but the readings were wrong, reading were not increasing as it supposed to be and the blank tube has the highest reading!
I repeated the same protocol twice. However, the results maintained the same!
Thank you all!
Dear Oumaima El Hachimi,
As you above written, the wrong is the volume of each concentration taken to assay, you must take different volumes by ml for each concentration that is equal different concentrations.
you can repeat the work with my advice and hope you the best.
Oday
Why did you add the galacturonic acid in about 10 times as much pectin? Probably the pectin has generated reducing ends after beta-elimination.
Is it possible the pectin itself might contain D-galacturonic acid and/or oligosaccharides? Have you done size exclusion chromatography on the pectin, and if so, is there a significant amount of material in the included volume (i.e. at the end of an SEC run where the small molecules elute? If you capture the included volume fraction and it turns out to contain reducing ends, then that's your answer.
I may say I would never prepare a standard curve of galacturonic acid where there was pectin present in the mix. The same goes for any polysaccharide and its monosaccharides: glucose and starch, glucose and xyloglucan, xylose and xylan, gluocosamine and chitosan, etc.
Hi dear Hachimi
it is method of Galacturonic Acid Standard Curve:
1. Prepare a stock solution of galacturonic acid 10 μmol /mL (Dissolve 48mg (250 μmol) of galacturonic acid in 25 mL of distilled water; (adjustable)
2. Place 1 mL each concentrations in a tube and add 1 mL DNS reagent; 3. Boil the content of all tubes for 5.0 min in a vigorously boiling water bath containing sufficient water to cover the portions of the tubes filled by the reaction mixture plus reagent. All samples, controls, blanks, and glucose standards should be boiled together; 4. After boiling, transfer to a cold ice water bath; 5. Dilute the content of all tubes (samples, blanks, standards and controls). Add 0.2 mL of color developed reaction mixture plus 2.5 mL of water in a spectrophotometer cuvette and mix well; 6. Measure absorbance at 540 nm; 7. Draw a standard sugar curve (sugar concentration along the x-axis vs. absorbance at 540 along the y-axis) following the volumes and standard concentrations of the enzyme assay. Different color intensities are generated by the different sugar concentrations. best regrades,
I would like to ask about the use of glucose as standard for pectinase assay?is it allowed?
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