# How do I calculate DPPH assay IC50?

After I plot % inhibition versus concentration (ug/ml) in excel. What do I do next to find out IC50? Please help!

After I plot % inhibition versus concentration (ug/ml) in excel. What do I do next to find out IC50? Please help!

## All Answers (35)

Barbara Tomasello· University of CataniaI hope that these tips will help you...further information on http://www.ncsu.edu/labwrite/res/gt/gt-reg-home.html

Good luck

B.

DeletedI plotted a scattered plot using %inhibition versus concentration (ug/ml). After i have the linear regression equation y= mx +b, what is the value of my y?

Concentration (ug/ml) --> x

25

50

75

100

and

% Inhibition --> y

4.06

23.74

29.00

31.78

How to i get the 50% number of the % inhibition values to put into the equation as y? I couldnt use mean or median..

Oyais Ahmad Chat· University of Kashmirwhen u see it draw a vertical line . The point where vertical line touches x-axis gives u IC50.

There is one more mathematical method will update u with ref.

Oyais Ahmad Chat· University of KashmirDeletedOyais Ahmad Chat· University of KashmirDeletedor

cant i use the "excel add-in" to calculate? http://www.sciencegateway.org/protocols/cellbio/drug/hcic50.htm

DeletedConcentration (µg/ml)

25

50

75

100

and

% Inhibition

2.28

6.39

6.07

6.89

I dont know what should i do with calculating IC50

Please help

Oyais Ahmad Chat· University of KashmirLogit Regression

Exp((1.759 * Log(X) -8.555)) / (1 + Exp((1.759 * Log(X) -8.555)))

R = 0.886481

EC50 = 129.6411 (0÷35.0719)

Angular Regression

Y = Sin(0.29 * Log(X) -0.695)^2

R = 0.919899

EC50 = 164.18 (60.0194÷246290.4)

Probit Regression

Y = CumNorm(0.931 * Log(X) -4.607)

R = 0.899136

EC50 = 141.1165 (44.2249÷1.410962E+21)

enjoyy!!!!

DeletedPeter Howgate· Independent ResearcherIf you want a reasonably accurate estimate of the IC50 you need to arrange your analyses so that the domain of your results includes 50% inhibition and the range of % inhibitions are approximately symmetrical around the 50% level. You then fit a logit or probit regression to calculate the IC50.

Barbara Tomasello· University of CataniaI could suggest Dep to add other experimental points (concentrations exceeding 100 ug / ml) .. so he can experimentally verify whether your data fit with those calculated by Oyasis..what do you think about?

Also, there is another problem .. he gets two completely different data sets with the same concentrations of substance: is impossible! .. However, the % inhibition values indicate that the substance is not a good antioxidant considering the selected concentrations.

But I can also think that perhaps Dep’s making a mistake during the pre-analytical phase-

Dep should check your protocol: vials, solvent , substance stability , blank sample

Mohammad Hossein Tavassoli-kafrani· University of AlbertaI observed this condition when I check an antioxidant, which it did not reach to the 50% inhibition in any concentration.

The regression for radical scavenging activity is exponential for most of antioxidants.

It should be added that Radical Scavenging activity do not tell the antioxidant activity and it should be use beside other tests.

Some times an antioxidant does not show a good radical scavenging activity but in other test such as oven test , shows a strong antioxidant activity since its main antioxidative activity is done by other mechanisms rather than radical scavenging .

good luck

Mohammad Hossein Tavassoli-kafrani· University of AlbertaThen your absorance must be between 0.3 - 0.7 (optimum) for your sample. if it not you should bring in this region by diluting or concentrating.

Oyais Ahmad Chat· University of KashmirSatyajit Kanungo· Saraswati Jr. Sc. College & Resonance Residential College, Cuttack, Odisha.The solution of DPPH was prepared by adding 4.3 mg of DPPH (1, 1-Diphenyl –2-picrylhydrazyl) was dissolved in 3.3 µl methanol; it was protected from light by covering the test tubes with aluminum foil. 150 µl DPPH solution was added to 3ml methanol and absorbance was taken immediately at 517nm for control reading. 50 µl of various concentrations such as 40μg/mL, 80μg/mL, 120μg/mL, 160μg/mL, 200μg/mL, 240μg/mL, 280μg/mL, 320μg/mL and 360μg/mL. of the plant sample extracts as well as standard compound (Ascorbic acid) were taken and the volume was made uniformly to 150 µl using methanol. Each of the samples was then further diluted with methanol up to 3µl and to each 150 µl DPPH was added. Absorbance was taken after 15 min. at 517nm using methanol as blank on UV-visible spectrometer Shimadzu, UV-1601, Japan. The IC50 values for each drug compounds as well as standard preparation were calculated. The DPPH free radical scavenging activity was calculated using the following formula:

% scavenging = [Absorbance of control - Absorbance of test sample/Absorbance of control] X 100

The effective concentration of sample required to scavenge DPPH radical by 50% (IC50 value) was obtained by linear regression analysis of dose-response curve plotting between %inhibition and concentrations (Iranshahi et al., 2009).

Atul UpadhyayAtul UpadhyayDeletedCurrently now, I only could calculate IC50 by input 50 in y for the linear equation, although it is not accurate. =(

Yogesh Kumar· Pondicherry Universityenjoy

Edith Oliva Cuevas-Rodriguez· Universidad Autónoma de SinaloaAlok Nahata· Dr. Harisingh Gour UniversityIf you are hopeless from all the analysis, Pls follow the old method. A bit time consuming but you will get the exact result. Use a graph paper to plot the curve between % inhibition and concentration. Then manually find the 50% inhibitroy concentration for each sample. It is simple and easy. Other wise you can use the Graph Pad Prism or Instats statistical softwares for this purposes. They also work well.

The manual method is also very well accepted by high impact international journals. So don't worry about acceptability of this method. Here I have shared my experience. haha

GUD LUCK

regards

DeletedCan i use IC10?

Yogesh Kumar· Pondicherry Universityit also depends which protocol you are following and what instrument you are using to take O.D.

Ajit Kumar· University of DelhiAtul UpadhyayAnupam Khiste· Narsee Monjee Institute of Management StudiesAnupam Khiste· Narsee Monjee Institute of Management StudiesRathi Sre· University of MadrasRathi Sre· University of Madras..

Mangai Alarmal· Karpagam College of EngineeringAbhishek Yadav· VIT UniversityAntonio Zuorro· Sapienza University of RomeHowever, as somebody suggested before, it is better to make an intrapolation, so you should have at least one point at a concentration where the IC is higher then 50%.

Usually, I have an excel sheet with an atomation for this calculation... if you need it, just ask me.

Rathi Sre· University of MadrasLydia awuor Ogonda· University of NairobiNote: this val;ue is only reliable where the R2 value of the curve is close to 1

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