Quick Guide to Calculating Enzyme Activity

Рет қаралды 127,531

Mark Temple

10 жыл бұрын

Using Excel to do the Lineweaver-Burk plot

Пікірлер: 46

@bronation9669 4 жыл бұрын

I spent over 3 hours trying to this myself only to find your awesome video. American student here thanking you across the globe for being an incredible teacher and furthering my scientific knowledge by posting this!

@gihchemistry 4 жыл бұрын

The best class I have had about this issue. Thank you!

@maddiebear2587 3 жыл бұрын

Best teacher. I have found this lovely video after I've already passed the this unit. Very late... but referring to my friend. Keep posting Dr Mark.❤

@mountcarmel7116 3 жыл бұрын

Thank you professor, you guided me to get out of many confusion.

@ahmadmustafa1597 10 жыл бұрын

Thank you professor, this is very helpful

@nasihaahmed8872 10 жыл бұрын

This was really helpful, thanks Mark

@ebenezerappiah5528 3 жыл бұрын

Hello Temple thanks for the incredible work you do for some of us struggling with some research procedure. God richly bless you. Please is it possible to get a link to the research article showed in this video. I have been searching for it online but cant seem to get exactly what you were using in this video. Thanks very much

@DrMuhammadAjmalShah 8 жыл бұрын

Hi Mark Temple, I am performing kinetic of alpha-glucosidase inhibition assay in which enzyme concentration is constant with varying concentration (5 ) of substrate (PNGp) and varying concentrations (3) of inhibitors (isolated natural compounds and positive control acarbose). I got absorbance reading for all samples and control @ 0.5 minute interval for 40 minutes. So how can I proceed now to calculate velocity which is required for plotting and then how to proceed for plotting Linear weaver burk to obtain the enzymatic inhibition mechanism for each sample. I need basic information because i never work before with enzyme

@g00dej 8 жыл бұрын

Dear Dr. Mark Temple, your online tutorial is really clear and useful!!! So, I used your data to practice calculating enzyme activity, everything is all right, but I'm not sure if the accurate result of Tube 5 of Dilution 1 in Table 2. is 0.1111111.

@user-yj4cb8gk4z 5 жыл бұрын

Dear Sir, Would you please help me in plotting the readings of time dependent GTPase assay using malachite green?

@TheForeverFalcon 4 жыл бұрын

Great Video, learnt a lot.

@sushmachauhan8103 6 жыл бұрын

Dear Dr. I would like to know how to calculate specific activity of enzyme. I am doing enzyme assay for Horseradish peroxidase and i have to calculate specific activity. I would be very helpful for me if you will explain me how to do.

@Marianasantos-xn6yj Жыл бұрын

amazing! thank you so much for this!!

@manuelvenegas195 5 жыл бұрын

Hello, this video is very helpful. Where can I get a hold on this exercise?

@tringuyencsk32 3 жыл бұрын

Dear Professor. If I can not calculate product forming, I just can identify disappearance of substrate concentration follow time. How can I calculate velocity? Thank you so much, Professor.

@user-nv1ge8bh6d 2 ай бұрын

thank you for your help mark

@zuhropuput8932 2 жыл бұрын

sorry professor, I want to ask you, what is the basic principle used in determining the initial rate (v0) ?

@abdurrahmanadam1967 2 жыл бұрын

Sorry may I ask why it has to be quadratic polynomial to to find the initial rate? Thank you

@pggroup8582 8 жыл бұрын

Dear sir, can you please give me the link for "the last week video" that you have pointed out in the beginning of the video. I have tried to search for it but could not find out. I have to study the enzymatic activity and have to do similar kind of experiments. It will be a great help. Kindly respond

@UWSMarkTemple 8 жыл бұрын

+Pg Group Early During in the video I refer to the "last week", this comment does not refer to another video but is a reminder that "last week" laboratory work was performed which generated the data. This data is subsequently analyses in the video. The experimental work consisted of making a standard curve for p-nitrophenol, and then running a series of reactions in which enzyme concentration is kept the same but substrate concentration was varied. This was done to establish how substrate concentration affects reaction rate. The reaction rate was assessed by monitoring the formation of product over time (3 min). The amount of product was estimated from the standard curve. Hope that helps.

@thoithoisanasoibam879 3 жыл бұрын

Sir I am studying inhibitory effect of plant extract on pancreatic lipase enzyme...I am not able to develop a protocol for it....could you please help me ...

@alaechda9056 Жыл бұрын

One enzyme unit (U) is defined as the amount of enzyme that transforms 1 µmol of substrate in 1 minute (µmol / min), should we transform absorbance to the amount of the substrate or to the amount of product to have U

@UWSMarkTemple 9 жыл бұрын

In response to "I'm an aspiring research student Philippines. And I'm currently doing my Research Paper to compare which sample has more enzyme activity. Im gonna use the fruit itself both ripe and unripe, the seeds, leaf and the roots. Now my problem is that the enzyme calculations on whether who has the greatest amount of enzyme activity present. But I have difficulty in understanding. Please help me. I will indeed be thankful if you support and help me. God bless you and I hope u will see my message." Response: The video looks at a purified enzyme activity with a substrate at different concentrations. You seam to be looking at different enzyme preparations with a substrate at the same concentration. I guess you would be best calculating the ‘specific activity’ of the enzyme, e.g. Amount (micromole) of product per time (minute) per amount (mg or ml) of enzyme sample, under specified reaction conditions. This will give a unit of product formation something like micromole/min/mg. Do control experiments also e.g. Negative controls with heat denatured plant samples or an extract from another seed/plant type. A positive control is also important e.g. a sample with maximum activity to show that the assay is working reproducibly. Good luck

@moonspark123 2 жыл бұрын

Dear professor. Can you kindly upload the work sheet of this calculations shown in this lecture? We can practice with these calculation, as formulas are embedded these. Thanks

@saidprado826 9 жыл бұрын

May I use to find Vo, a linear (y=mX+b) better than a polinomial?

@UWSMarkTemple 9 жыл бұрын

Said Prado If you expect the plots to be a straight line then certainly use y=mx+b, this is true for the standard curve and the final Lineweaver-Burk plot. For the plots of the initial rate of reaction, these are not expected to be straight lines, particularly since these reactions take a finite time to kick off and tend to level off after a time. For these plots, i.e. the initial rates of reaction, technically you should use a polynomial curve to fit these data.

@saidprado826 9 жыл бұрын

i really appreciate your help thank you very much... by the way... so nice to meet you.

@avogadro871 7 жыл бұрын

Which order in polynomial should fit the plots of initial rate?

@UWSMarkTemple 7 жыл бұрын

The initial rates are quite linear to start with, you could approximate things by drawing a straight line through the first minute (or over a one minute period) and use the gradient of this line (rise over run) to approximate the initial rate. This would only require graph paper and not plotting the graphs on excel. Excel does not understand enzyme kinetics and often over complicates the simple things!

@mountcarmel7116 3 жыл бұрын

would you please explain how you got back to your standard graph to calculate the absorption into the amount instead of using the Beer lambert law?

@UWSMarkTemple 3 жыл бұрын

The standard graph shows how the absorption changes as the concentration increases. The beer lambert law states that this should be a linear relationship. Hence we draw a straight line through the data points. We can then take absorption values from later experiments using the same compound and read off the amount of compound it relates to. You could use A=ecl to calculate things but often you may not know the molar extinction coefficient (e) when researching something.

@anjusubramannian6781 5 ай бұрын

@@UWSMarkTemple😅

@vijaykumar-wz1vb 3 жыл бұрын

Sir, Can you explains how to convert absorbance into international unit

@viboluno3563 7 жыл бұрын

Dear Dr. Mark Temple. I have doubt, where X is multiplied by 10? It should be X multiplied by 10 and divided by 1.5 instead? after that why did you multiply it by 4? Thank your for your answer!

@UWSMarkTemple 7 жыл бұрын

Yes you are correct, we multiplied by 10 due to the difference in volume of the 0.150 ml and 1.50 ml tubes, ie we have 10 times as much in the larger 1.50 ml tube (cuvette). But... in this larger 1.50 ml tube there was only 0.25 ml of the original enzyme and we want to know the specific activity of that original solution, hence in 1 mL of the original enzyme solution there would be 4x as much activity. (4x 0.25 = 1 ml). Its a bit confusing due to the actual sample manipulations at the bench. Hope that helps.

@viboluno3563 7 жыл бұрын

Thank you, professor.

@salmanah2518 4 жыл бұрын

Is it possible through the activity of the enzyme to find the concentration of the enzyme itself?

@UWSMarkTemple 4 жыл бұрын

1 mg of Enz1 may be 100x more active than 1mg of Enz2, therefore the number of mg/ml (concentration) is independent of the activity. To get around this issue you need to consider the specific activity of the enzyme - this measure/unit takes into account both the amount of enzyme and the activity of the enzyme. If you were dealing with a crude extract of EnzX then as it becomes more purified its specific activity would increase.

@tsiporacohen-taieb2195 3 жыл бұрын

Are we can calculate the enzyme activity without a standard curve ?

@UWSMarkTemple 3 жыл бұрын

The STD curve was made using dilutions of a solution that contained a known amount of compound. Therefore the absorbance values can easily be related to the amount or concentration of that compound. So when you later measure the absorbance of an unknown containing that compound you can use the STD curve to map back to the amount/concentration in the standard. Alternatively you could lookup the extinction coefficient (e) of the compound if it is known and use Abs = e x [conc] x lightpath (i.e. A =ecl ). All you want is to determine how much of the compound is being made by the enzyme reaction and at what rate. From there you can go ahead and think about the activity of the enzyme, how much enzyme was in the tube and how active was it to make/release the compound you measured.

@tsiporacohen-taieb2195 3 жыл бұрын

@@UWSMarkTemple thank you !

@RameshKumar-cm1cg 7 ай бұрын

❤❤

@user-si9xi8mc3p 4 ай бұрын

oh you gorgeous gorgeous man thank you so much

@MuzaffarJan Жыл бұрын

why is eqn y=mx-c used here. it should be y=mx+c

@hamedzaid1699 3 жыл бұрын

I wish I had access to these movies and descriptions last semester so at least I could learn something.. I don't want to name him but the worst ever class and teacher I had in my entire life. He used to just memories things and then come and telling us without proper description. He didn't know and describe things and concepts properly.. I wish I could provide feedback about him.