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Topic: HI pls help in this enzyme activity assay  (Read 5233 times)

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Offline tedanton

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HI pls help in this enzyme activity assay
« on: August 31, 2009, 11:53:18 PM »
I am going to do a new lab for undergraduates on enzyme activity. Themain aim of  this lab is that the students are expected to get a standard curve once they finish the assay. NAGase(20 microlitre in 120 ml of Citrate buffer ,4.5 pH and 20 mg BSA) is the enzyme used to measure the activity.This is then diluted to different con .Susbstrate  is NAG(70 mg in 100 ml of citrate buffer).These are then mixed together and incubated for 15 mts at a temp of 50 degree.spectrophotometer readings would show absorbance of 0.7 over the period of 15 mt , if things go well. since I am doing it for the first time, I tried a trial before the main lab. There was no reaction happening when i did the assay .What could have gone wrong with the experiment. I need your valuable advice

Offline Yggdrasil

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Re: HI pls help in this enzyme activity assay
« Reply #1 on: September 01, 2009, 12:03:03 AM »
NAGase(20 microlitre in 120 ml of Citrate buffer ,4.5 pH and 20 mg BSA) is the enzyme used to measure the activity.This is then diluted to different con .Susbstrate  is NAG(70 mg in 100 ml of citrate buffer).These are then mixed together and incubated for 15 mts at a temp of 50 degree.spectrophotometer readings would show absorbance of 0.7 over the period of 15 mt

20µL in 120mL seems like a very dilute solution.  Do you mean 20µL in 120µL?  Also, 70mg in 100mL buffer also seems very dilute.  Also, what does NAG stand for (is it N-acetylglucosamine)?

Do you have a reference to any published work on the enzyme?

Offline tedanton

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Re: HI pls help in this enzyme activity assay
« Reply #2 on: September 01, 2009, 12:16:21 AM »
no, its 20 micro litre  in 120 ml ,this is done so as to get an activity of  4 units /ml. from this stock solution of (4 units/ml ) , series of different con are prepared(4,3,2,1.5,1,.5,.2,) by diluting with the same citrate buffer to 20 ml.Nag stands for N acetyl glucosinamide.

Offline tedanton

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Re: HI pls help in this enzyme activity assay
« Reply #3 on: September 01, 2009, 12:25:58 AM »
Nagase is the enzyme realeased by the damaged secretory cells in the mammary glands of cows with mastitis.The amount of nagase in milk will tellabout the infection in mammory gland

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