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Conversion of Absorbance into enzyme units


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#1 San1986

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Posted 05 August 2012 - 06:49 AM

Hi all,

I'm working on cellulases from bacteria. I've taken O.D after several parameters to check the enzyme activity. I'm using Sodium Acetate buffer of pH 5 (0.1 M) in my enzyme assay. I take 0.2 ml crude enzyme, 0.5 ml substrate buffer (substrate prepared in Acetate buffer of pH 5 (0.1M)) ad 0.3 ml of Acetate buffer as stated above. It makes up the reaction mixture 1 ml in total. I incubate it at 50 C for 15 mins, then add 3 ml DNS and then boil for 5 mins, cool down and take the Absorbance. For example, if Spec give the reading as 0.375. How would i convert it into enzyme units?

I have tried to search it on internet but couldnt find anything understandable.

Thanks in advance :)

San.

#2 bob1

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Posted 05 August 2012 - 01:54 PM

YOu can't unless you have a standard (cellulase of known concentrations) to compare it to.

#3 San1986

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Posted 06 August 2012 - 11:44 AM

I will make up a Glucose standard curve with known concentrations. Should i post the standard curve to know the formula?

#4 bob1

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Posted 06 August 2012 - 04:44 PM

YOu can get the formula from most graphing programs. I recommend Graphpd Prism, but even excel will do the job.

#5 San1986

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Posted 07 August 2012 - 10:55 AM

I cant find how would i use excel for this? Should i plot both the curves (the standard and the one which is different absorbances of the enzyme) over each other?

#6 bob1

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Posted 07 August 2012 - 01:57 PM

Normally you would plot absorbance on the y axis and concentration of the standards on the x axis(in any graphing program, but if you have excel, you can use that). The following instructions are for excel:Highlight your data, and choose "insert chart", the option you need to choose is a scatter plot Once you have a line, you can get a best fit by right clicking on a data point on the chart and choosing "add trendline". Choose the type of line that it is (straight, quadratic, etc.) and check the "r squared" and equation boxes.

#7 San1986

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Posted 09 August 2012 - 09:15 AM

Thanks bob. I finally explored an option which i didnt know. :)




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