I have been using 10g NaoH, 10 g DNS, 2g Phenol and 0.2 g Rochelle salt. Later Sodium sulfite is being added to the stock solution while preparing the working solution. But dark brown to black color develops for 0.8 and 1.0 mg/ml xylose and its OD goes beyond measurable level at 575 nm.
You can follow the procedure provided in publication and link.
http://www.eng.umd.edu/~nsw/ench485/lab4a.htm
Article Adapting the Reducing Sugars Method with Dinitrosalicylic Ac...
Thanks for sharing the paper. I was earlier using the protocol by Nam Sun Wang (same as the link provided by you). But color develops beyond the measurable level.
It may be that 0.8 and 1.0 mg/mL Xylose is too concentrated and needs to be diluted for it to be in the range for measurement.
i so confused about protocol for DNS. Some of them just used only 3-5 DNS and sodium potassium tartrate. Another is mix with phenol, NaOH and Na metabisulfite as well. What are different between these.
I'm using the same protocol. i didn't get any color. It's crystal clear.
Preparation steps of 3,5-dinitrosalicylic acid solution 100(ml):
1. Dissolve 30(g) of Potassium sodium tartrate tetrahydrate in 20(mL) distilled water. Add the salt to water gradually.
2. Prepare 2(N) sodium hydroxide solution 20 (mL). Dissolve smoothly sodium hydroxide in water using a glass rod and stirring the mixture of granules in water.
3. Dissolve 1(g) of 3,5-dinitrosalicylic acid in 50 (mL) of distilled water while the solution is mixed by magnetic stirrer with hot plate at 90-95˚C.
4. Add gradually the solution of potassium sodium tartrate tetrahydrate (prepared solution in Step 1) to 3,5-dinitrosalicylic acid solution (prepared solution the Step 3) while the solution is mixed by magnetic stirrer with hot plate at 90-95˚C.
5. Add slowly 2(N) sodium hydroxide solution (the solution prepared in step 2) to the solution prepared in step 4 while the solution is mixed by magnetic stirrer with hot plate at 90-95˚C.
6. After the components are completely dissolved, filter the final solution by filter paper.
7. Transfer the solution in dark glass bottles and storage at ambient temperature.
For estimation of reducing sugars using DNS :
1. Prepare the sugar solution according your favorite concentration
2. Prepare phosphate buffer solution (o.1(M), pH=7)
3. Add 1(ml) of sugar solution to the test tube then add 2(ml) DNS solution (it can be considered as a main sample)
4. Add 1(ml) enzyme extract to test tube then add 2(ml) DNS solution (it can be considered as a blank or control sample)
5. Transfer the tube to the water bath at boiling temperature for 15(min)
6. Keep the test tube at room temperature to cool the sample
7. Transfer the solution to the cuvette
8. Measure the absorption of the main sample at λ=540(nm)
It is necessary to mention that this method is used when there is not any enzyme which produce reducing sugar. According to the type of enzyme the method must be changed.
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