How to prepare 10 ppm p-nitrophenol solution?
Dilute 1 mL of p-nitrophenol solution to 100 mL, it contains 10 ppm p-nitrophenol solution. 2. Pipette out 0,1,2,3,4, and 5 mL of 10 ppm p-nitrophenol solution in 50 mL volumetric flask and add 5 mL distilled water.
How to prepare p-nitrophenol standard curve while measuring phosphatase activity?
The following steps we are following for preperation of p-nitrophenol standard curve while measuring phosphatase activity. 1. Dilute 1 mL of p-nitrophenol solution to 100 mL, it contains 10 ppm p-nitrophenol solution.
How to prepare 10mm PNP stock solution?
> 10mM pNP stock solution is prepared by dissolving 0.0139 g of pNP in 10 ml of desired buffer. > Working stock solution of 1mM pNP is used for preparing std. curve. > A pNP concentration range of 0 to 100µM (or 0 to 0.1 mM) is used by making appropriate dilutions from the working stock (preferably in 1ml total volume).
How can I establish a PNP standard curve?
I have established pNP standard curve by taking pNP dilutions 0 to 1 mM (1 µmol/mL) in 20mM tris (pH ~6, optimum pH of my enzyme too). I added 1M sodium carbonate (Na2CO3) to all the samples in the standard range including blank.
How do you calculate PNP concentration?
1 AnswerCalculate the concentration of PNP before dilution. Moles of PNP=1.25 mg PNP×1000 µg PNP1 mg PNP×1 µmol PNP139 µg PNP=8.99 µmol PNP. ... Calculate the concentration of PNP after dilution. You added 12.0 µL to 488 µL, so your new volume was 500 µL.
How is Vmax calculated?
The rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction, Vmax....plotting v against v / [S] gives a straight line:y intercept = Vmax.gradient = -Km.x intercept = Vmax / Km.
How do you calculate units of enzyme in ml?
Enzyme units are expressed as µmol substrate converted per min. If the question gives enzyme activity in nmol per min, divide by 1000 to convert to µmol. Then multiply by the volume to get the total number of units.
How do you calculate substrate concentration?
1:3610:02Michaelis Menten equation - YouTubeYouTubeStart of suggested clipEnd of suggested clipNow the first-order reaction kinetics can be explained easily with the equation y is equal to MXMoreNow the first-order reaction kinetics can be explained easily with the equation y is equal to MX plus C where Y is the velocity. And mr. Slope C is the intercept on y-axis.
What is Vmax?
Vmax is the reaction rate when the enzyme is fully saturated by substrate, indicating that all the binding sites are being constantly reoccupied. From: Introduction to Biological and Small Molecule Drug Research and Development, 2013.
What is Vmax measured in?
moles/minThe Vmax unit is moles/min, moles/sec, µmoles/min, or µmoles/sec. Vmax depends upon the amount or the concentration of the enzyme as well as the structure of the enzyme. Biology definition: Vmax is the maximum initial velocity or rate of a reaction.
How do you calculate enzymes?
Enzyme activity = moles of substrate converted per unit time = rate × reaction volume.
What is units per mL?
The results of some medical tests are reported in units per millilitre (U/mL). A unit is an arbitrary amount agreed upon by scientists and doctors. A millilitre is a unit of fluid volume equal to one-thousandth of a litre. A litre is slightly larger than a quart.
How is enzyme concentration calculated?
For example, if the enzyme concentration is 0.147 µg/µl, and the specific activity is 152 pmol/ µg min, the enzyme concentration is: 0.147 µg/µl x 152 pmol/ µg min = 22 pmol/ µl min = 22 nmol/ min ml = 22 U/ml. It needs to be diluted 1: 22 to achieve 1U/ml.
What is concentration of substrate?
Substrate concentration is the amount of substrate present that can be turned into product and is most commonly measured in molarity (moles per liter). The concentration of substrates is often used to measure enzyme activity, which is based on the rate of a reaction (product formed over time).
How do you find the substrate?
One way to identify potential protease substrates is to determine the peptide sequences they cleave in vitro, in other words, which amino acids span the cleavage site and are recognized by the enzyme's active site. These sequences are then used, like partial license plate numbers, to search the proteome for substrates.
How do you calculate substrate concentration from absorbance?
The equation should be in y=mx + b form. So if you substract your y-intercept from the absorbance and divide by the slope, you are finding the concentration of your sample.
Most recent answer
Shuddhodana .. Thanks for sharing your protocol to generate pNP std. curve. I measured abs at 405, 410, and 420 nm by using 0 to 0.1 mM pNP. I got the best curve and regression at 420 nm. I am using 20mM tris buffer (pH~6) which is optimum for my enzyme. I have reviewed many good articles which recommend 405 or 410 nm absorbance.
All Answers (7)
Attached please find a Word file containing the procedure to make PNP standard curve.
Most recent answer
There are too many variables in doing the standard curve to be concerned about the very slight deviation that you observed. For example, how accurate is the concentration of your solutions? How precise is the volume your pipettor is delivering? There's an expression- don't let perfect be the enemy of good enough.
Popular Answers (1)
You need to know the the extinction coefficient (epsilon: e) of your product then you apply the Beer Lambert Abs= e c l (l is the pathlength if you use cuvette of 1 cm then you can calculate c (concentration of product that appeared or substrate that disappeared) by Abs/el . Be careful with the units of e, to determine the C (usually in mM).
All Answers (42)
You need to know the the extinction coefficient (epsilon: e) of your product then you apply the Beer Lambert Abs= e c l (l is the pathlength if you use cuvette of 1 cm then you can calculate c (concentration of product that appeared or substrate that disappeared) by Abs/el . Be careful with the units of e, to determine the C (usually in mM).
What is a solution?
Tap card to see definition 👆. A solution is a homogeneous mixture of two or more substances. Solutions are common - Most of the liquids and gases we encounter every day are actually solutions. A solution has at least two components. Click again to see term 👆.
Does nitrogen bubble out of a solution?
The solubility of most solids decreases with decreasing temperature. However, the solubility of gases increases with decreasing temperature. Therefore, the nitrogen becomes more soluble and will not bubble out of the solution.