I am doing experiment to produce alpha glucosidase from thermophilic bacteria. Though I can determine enzyme activity by enzyme assay but my supervisor wants me to have a positive control, in this case, comparing activity of crude enzyme extract to the standard alpha glucosidase (from Merck). But the problem is I cannot decide which amount of standard enzyme powder to do the assay. And I think it's not equivalent to compare them since the crude extract is likely to contain many contaminant proteins.
Is there any suggestion for me? Any clues will be valuable for me.
Thank you for reading my question!
Hi there,
What your supervisor wants is to calibrate your assay (activity versus mass of enzyme in the assay) in order to determine how much of enzyme you have roughly in your crude extract (provided contaminants have no effect on the enzyme activity). You should have the specification of the Merck enzyme (the specific activity which makes the link between activity and the amount of enzyme). Knowing this you should be able to determine what amount of enzyme to introduce in a standard assay to observe the expected velocity of reaction.
Hi there,
What your supervisor wants is to calibrate your assay (activity versus mass of enzyme in the assay) in order to determine how much of enzyme you have roughly in your crude extract (provided contaminants have no effect on the enzyme activity). You should have the specification of the Merck enzyme (the specific activity which makes the link between activity and the amount of enzyme). Knowing this you should be able to determine what amount of enzyme to introduce in a standard assay to observe the expected velocity of reaction.
Hi Phoebe,
a positive control just shows that your assay works and is able to determine the activity of an alpha-glucosidase meaning you do everything correct. If the Merck alpha glucosidease is exactly the same enzyme, you can prepare a standard curve to determine the amount of enzyme in your crude extract. If it is not, then this is not possible. you need to take a diluted amount of Merck enzyme that can be measured with your assay meaining it has to be in the linear range.
Hi Martin Lehmann
Thank you for giving me a very useful answer.
My supervisor also talked the same as you. But Im not clear about making "standard curve to determine the amount of enzyme in your crude extract".Did you mean I need to serial dilute Merck enzyme and make the standard curve? Then based on the curve I can get the idea about how much activity my crude enzyme has?
I
First of all the Merck enzyme should be the same/identical enzyme (from the same organism, same amino acid sequence) you like to find in the crude extract, otherwise this does not make so much sense. Then you have to define the enzyme concentration range, for which you assay shows linearity. You dilute the Merck enzyme to desired concentration (4 to 5 different concentration of the intended range) and plot measured OD (y axis) against used enzyme concentration in U/ml or mg/ml. With sample/crude extract OD you go into the standard curve to calculate the corresponding U/ml or mg/ml of Merck alpha-Glu.
Please follow what Martin has communicated to you. The specific Product at least from MilliporeSigma a subsidiary of Merck is G6251. It is relatively crude itself as it is >= 50 Units/mg protein and is supplied as a 50% protein powder. It as well contains up to a maximum of 0.1% Beta-glucosidase and Alpha and Beta Galatosidase. To compare you can always obtain the Assay procedure from MilliporeSigma to make sure it is assayed in the same manner.
Martin's answer is perfect. You need to do standard curve from time to time in order to check accuracy of your reagents and methodology. For this, you need to preserve standard enzyme properly. Make its small aliquots. Decide the frequency of making standard curve based on frequency of making new reagents/new kits.
Thank you @Ashvini Shete. I have followed Martin's instruction and it worked!
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