Hii...Nizzal Syafiq..

For the estimation of reducing sugar using DNSA method, you have to prepare standards curve of Glucose along with the your test samples in triplicates. Based on, standard graph, calculate R2 value (should be >0.9) and mg of glucose released under standard conditions and then calculate the enzyme units per gram of samples or ml.

Best wishes...

TQ Mr Yogesh for your answer.

Do you know why do we have to conduct the preparation of glucose standard graph along with the DNS assay of the enzyme sample? wouldn't it be easy if we can do both separately?

Nizzal, glucose standard graph is prepared as reference for DNS assay only in case of those enzymes where the end product of the reaction is glucose or reducing sugar. By preparing the standard graph you can calculate the performance of enzyme with respect to substrate hydrolysis (starch in case of amylase where the final end product is glucose, cellulose in case of cellulose where the end product is again glucose). More is the end product released/ ml/ min by the enzyme, better is the enzyme activity. Hope I could answer to your question and have clarified your doubts to some extent.Best regards: Deepti

TQ Dr Deepti for your explanation on the matter.

What I intended to know is can we do the standard graph preparation first before the DNS assay of enzyme sample? to be specific can I do standard graph today and for the next day I conduct the DNS assay of my enzyme sample.

I've heard from my lab mates that both have to be conduct at the same time because if we conduct it separately the result would not be so specific due the the different condition of both set. (which is personally i think it doesn't matter because the microclimate difference would not significantly matters)

Not necessarily.... If the DNS stock solution is the same. The standard glucose will give more or less same readings every day. It would not be affected so drastically. The y= mx+ c value or the slope of the graph will remain more or less same ( less than 0.5% standard error)

Nizzal Syafiq,

It is ideal that both standard glucose (reference) and test samples should be performed simultaneously to avoid variations in the results due to the experimental errors..Some people generally use the previously measured R2 value to calculate the reducing sugar but it is not recommendable..!!

It save time if we prepare our standard curve first before the sample analysis. But my suggestion is if you prepared new reagent then you have to prepared standard curve accordingly and if your experiments extended up to 2-3 months then it is wise to do standard curve in between them freshly and took the regression line for the determination of glucose concentrations for activity determination. Hope you will be benefited by this. All D Best.

-AKM

TQVM to Dr Deepti, Mr. Yogesh and Dr. Abhas for your well-explained answer for my question.

What I can conclude from the discussion above it would be depends on factor such as the freshness of DNS reagent. But somehow to those who concern on the tiny detail on the research would prefer to prepare the standard curve along with the test sample. Now I get the real picture on this matter.

TQ again for your fruitful discussion. Best wishes.

Best wishes Nizzal Syafiq..!!