Hi. I am currently working on alpha amylase inhibition assay using DNS reagent.
As the first step, I am preparing the DNS reagent and testing it with glucose to ensure that the DNS reagent is prepared the correct way.
After boiling the mixture of glucose and DNS reagent, I don't see any change in the colour of the mixture.
I prepared DNS reagent using the following steps:
Dissolve 1g of 3,5 dinitrosallicylic acid in 20mL 2M NaOH. Then add slowly 30g sodium potassium tartrate and dilute to a final volume of 100mL using distilled water.
Please advice on the procedure to prepare DNS reagent. thank you
• Colour Reagent: 96 mM 3,5-Dinitrosalicylic Acid (DNSA) with sodium potassium tartrate solution
- Slowly dissolve 2.18 g of DNSA (MW=228.1 g/mol) in 80 mL of 0.5 M NaOH by heating and stirring at 70º C.
- Add 30 g of sodium potassium tartrate (MW=282.2 g/mol) and stir until dissolve.
- Cool to room temperature and bring to 100 mL with purified water
May be trying this recipe…
Dissolve 10g of 3,5 dinitrosallicylic acid in 200mL 2M NaOH. (1gr-20 mL)
Dissolve 300g of sodium potassium tartarate in about 500 ml distilled water. (60g-100mL).
Mix 1+2, final vol 1L (f.v. 150 mL).
0.1g anhydrous glucose is dissolved in distilled water and then raised the volume to 100 ml with distilled water.
After mix reagent and glucose solution shake well and then place them in a boiling water bath for 5 minutes.
Cool the tubes thoroughly and then add 7 ml of distilled water to each tube. Read the extinction of the colored solutions at 540 nm using a blank (control), (1 mL of distilled water + 1 mL de DNS).
All the tubes must be cooled to room temperature before reading since the extinction is sensitive to temperature change.
You need the folowing
1. 1g of Dinitrosallicylic Acid
2. 30g of Sodium Potassium Tartarate
3. Sodium Hydroxide (2M) 20mls
So, to prepare 100mls of DNSA, you will need to mix 30g of Sodium Potassium Tartarate with 1g of DNSA and 20mls of NaOH. Then you make it up to 100mls with distilled water. You can scale it up if your assay demands it and you can still scale it down as well.
• Colour Reagent: 96 mM 3,5-Dinitrosalicylic Acid (DNSA) with sodium potassium tartrate solution
- Slowly dissolve 2.18 g of DNSA (MW=228.1 g/mol) in 80 mL of 0.5 M NaOH by heating and stirring at 70º C.
- Add 30 g of sodium potassium tartrate (MW=282.2 g/mol) and stir until dissolve.
- Cool to room temperature and bring to 100 mL with purified water
i would like to know why is Sodium Potassium Tartarate added ???
Sodium Potassium Tartarate also known as Rochelle salt.
hope the pdf file will help
Be careful if you use the Na K tartrate x4H2O (tetrahydrate). You must calculate the quantity of NaK tartrate without H2O. For Mehmet Akif: you must have a final volume of 100 ml after adding the tartrate. How do you think to prepare the 100 ml of dns reagent and after the addition of tartrate the volume is the same?
Best way to prepare DNS reagent is to use IUPAC or NREL protocol step by step. you can then use IUPAC protocol for glucose standard preparartion and modify it to 1 ml reaction instead of 1.5 mL and add glucose working standards in such a way that final reaction contains 0.1 mg to 1 mg (per mL). Boil for 5 min in a boiling water bath with sufficient water. Chill the tubes 1-2 min in ice bath or ice-cold water and then Add 20 mL DW and measure OD at 540 nm
NaOH solution is supposed to be 2N by concentration, not 2M. (Normality, not Molarity) For 2N NaOH solution, you can simply dissolve 8.0 gram of NaOH in 100.0 ml distilled water..
And DNS reagent preparation can be briefly dissolving 1.0 gram of DNS in 20 ml 2N NaOH and adding 30.0 gram sodium potassium tartrate to a final volume of 100.0ml with dH2O. Your protocol is not that wrong.
Please can any one answer this question
https://www.researchgate.net/post/What_about_alpha_amylase_concentration_for_the_enzyme_inhibition_test
I would like to correct Müge, for NaOH molarity and normality are same; M=N. In some cases, like H2SO4, normality differs from molarity, in this case 1M = 2N
Dear Sutha Muniandy,
For 100 ml of DNS reagent,
i) Take 1g NaOH in 70 ml distilled water, mix well.
ii) Add 30 g of Potassium Sodium Tartarate to it and mix till it dissolves fully.
iii) Add 1 g of DNS powder, continuously stir till it dissolves.
iv) Add 0.05 g of Sodium Thiosulphate to it and dissolve fully.
v) Add 0.2 g of Phenol crystals.
vi) Make up the volume to 100 ml.
Your DNS reagent is ready. Preferably prepare it freshly for better results or store it in a cool and dark place and use it within 3-4 weeks.
Hope this helps you,
Thanks.
Instead of giving answer, i also want to know, what is the different between adding tartarate into DNS reagent or, make it separately and added when it use to glucose sample
Dissolve 10 g of 3,4-dinitrosalicyclic acid in 200 ml water in 1000 ml beaker. Followed by continuous stirring slowly add a solution of NaOH dissolved in 150 ml distilled water. Incubate mixture in 50°C with stirring to obtain a clear solution. In small portions add 403 g of potassium sodium tartrate tetrahydrate. Filter mixture using paper filter and make up the volume to 1000ml with water. Store in dark glass bottle at temperature below 20 °C. This has to be used within 20-30 min.
Just wondering how would I dispose off the DNS reagent then? Can it just simply go down the sink?
Hope this helps you ............
(NaOH solution is supposed to be 2N by concentration, not 2M. (Normality, not Molarity) For 2N NaOH solution, you can simply dissolve 8.0 gram of NaOH in 100.0 ml distilled water.. )
Sutha Muniandy
Hi Sutha Muniandy,
To prepare 100 mL of DNS (3 5-Dinitrosalicylic acid) reagent:
1). Add 1g NaOH to 70mL of sterile dH2O in a 250mL beaker, under constant stirring on a magnetic stirrer.
2). Add 30 g of Potassium Sodium Tartarate tetratehydrate, allow it to dissolve fully.
3). Add 1.06 g of DNS, allow it to dissolve fully.
4). Add 0.83 g of Sodium metabisulphite, allow it to dissolve fully.
5). Add 0.2 g of Phenol crystals.
6). Transfer the solution to a 100mL volumetric flask and make up the volume to 100mL with sterile dH2O.
7). Transfer the solution into a brown bottle.
8). Your DNS reagent is ready for use.
Cheers and good luck!
I create the reagent with this method:
1). Add 1g NaOH to 70mL of sterile dH2O in a 250mL beaker, under constant stirring on a magnetic stirrer.
2). Add 30 g of Potassium Sodium Tartarate tetratehydrate, allow it to dissolve fully.
3). Add 1.06 g of DNS, allow it to dissolve fully.
4). Add 0.83 g of Sodium metabisulphite, allow it to dissolve fully.
5). Add 0.2 g of Phenol crystals.
6). Transfer the solution to a 100mL volumetric flask and make up the volume to 100mL with sterile dH2O.
7). Transfer the solution into a brown bottle.
but the color was crystal clear instead of yellow. and i didn't get any color after boiling 5 min in boiling water. I don't know what was the problem.
DNS reagent:
Solution A:
8g NaOH
5g DNS (3,5-Dinitrosalicylic acid)
Aqua dest. to 100 ml
Solution B:
150 g KNa (Potassium sodium)-tartrate
Aqua dest. to 250 ml
Mix solution A slowly into solution B and fill with A. dest. to 500 ml. Heat up to over 100°C and keep stirring until disolved!
step 1: DNS preparation
1. add 2g NaOH in 200ml of H20
2. stir it completely until it dissolves
3. add 2g DNS and cover it immedietly
4. add 400mg of Phenol (400mg /density of phenol = .373ml )
5. phenol is melted at 50C for 30 min
6. add 100 mg of sodium sulphate is added
7. stir it completely until it dissolves
8. store it in fridge
step 2 : Preparation of Rochelles salt
1. add 8g of sodium pottasium tartarate tetrahydrate in 20ml water
step 3: Sample preparation
1. add 1.5ml of diluted sample + 1.5ml DNS in test tube
2. boil water in a beaker 100C
3. keep the test tube in boiled water for 5 min take it out and add 0.5ml rochell solution
4. keep the test tube in ice bath for 5 min
step 4 : OD measurement wavelength 540nm
1. blank solution 1.5ml water + 1.5 ml DNS prepared
How to store the prepared DNSA reagent and How long it can be stored ??
Please see this Link:
http://www.ncbe.reading.ac.uk/MATERIALS/Enzymes/dnsareagent.html
- Badges
- Science method
- Similar topics
- Chemistry
- General Biochemistry
- Biochemical Techniques
- Analytical Chemistry