The Quest Graph™ IC50 Calculator attempts to model an experimental set using a four parameter logistic regression model, so called because it has four key parameters in an equation of the form:

Y = Min +

Max − Min

1 + (

X

IC_{50}

)

^{ Hill coefficient}

This model typically resolves as a sigmoid function, or "S"-shaped curve. For biological promotion, the Hill coefficient of the equation will be (-) negative, with the slope of the curve rising ("S"). For biological inhibition, the Hill coefficient of the equation will be (+) positive, with the slope of the curve falling ("Ƨ").

With regards to IC50, the sigmoid function itself is a special case of the log-logistic distribution, which is part of a broader family of logistic distributions and functions. In contrast to standard logistic distributions, however, a primary distinction of this calculator is that it does not necessitate prior normalization of data, nor does it enforce these boundaries in the modeling of an experimental set. That is to say, in a standard logistic distribution, the response values (Y) range from 0 to 1 probability values. This is typically what is seen in probit/logit analysis and what is commonly used when modeling population survival rates. In this calculator, response values can be any positive real number, which may result in regression models which do not adhere to the 0 to 1 logistic distribution boundaries. This is especially the case where the controls of an experimental set do not make clear the upper and lower bounds of the data. In such cases, it is not uncommon to generate a regression model which extends far into the negative Y-axis or rises exponentially.

Because negative response values are typically incoherent in biological contexts, it is often desirable to analytical restrict regression models to the positive response domain. This can be achieved computational by fixing the minimum response value to zero. This reduces the four parameter logistic model into a three parameter logistic model with the simplified equation as follows:

Y =

Max

With this form, the regression model has a lower-bounds fixed at zero, eliminating any models which may otherwise extend into the negative response domain.

There are two final points to make about this calculator. The first is that the four parameter logistic curve is a symmetric regression model around the inflection point, that is, the IC_{50}. Roughly translated, this implies that the shape of the sigmoid function on one side of the inflection point will mirror that of the other side. In order to model an experimental set which is asymmetric in nature, a five parameter logistic curve is required. Second, it is inadequate to utilize R^{2} when discussing the goodness of fit of a four parameter regression model. As has been demonstrated in several papers, R^{2} can be used when describing linear regression models, but fails to capture the degree of noise and variability in non-linear models.

For additional reading, please see the following:

- Finney, David John, and F. Tattersfield.
*Probit analysis*. Cambridge University Press; Cambridge, 1952. - Sebaugh, J. L. (2011). Guidelines for accurate EC50/IC50 estimation.
*Pharmaceutical statistics*, 10(2), 128-134. - Spiess, A. N., & Neumeyer, N. (2010). An evaluation of R 2 as an inadequate measure for nonlinear models in pharmacological and biochemical research: a Monte Carlo approach.
*BMC pharmacology*, 10(1), 6.

Have a question or a feature request about this tool? Feel free to reach out to us and let us know! We're always looking for ways to improve!

## References

### This online tool may be cited as follows

## MLA | "QuestGraph™IC50 Calculator."AAT Bioquest, Inc.,2Sep.2022,https://www.aatbio.com/tools/ic50-calculator. |

## APA | AAT Bioquest, Inc. (2022,September2).QuestGraph™IC50 Calculator. AAT Bioquest.https://www.aatbio.com/tools/ic50-calculator. |

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## FAQs

### How is IC50 value calculated? ›

To calculate the IC50 value, **from the equation of the graph that you got (Y = MX-C), change Y to 50 (50= MX-C) then make X the subject of the formula**. The X value should be your IC50 value.

### What is a good IC50 value? ›

...

3.2 Determination of EC

_{50}/IC

_{50}/CC.

_{50}.

IC _{50} or CC_{50} | Criteria | |
---|---|---|

Isolated compound | Extract | |

< 1 μM | – | Excellent or potent activity |

1–20 μM | < 10 μg/mL | Good activity or very strong cytotoxicity |

20–100 μM | 10–50 μg/mL | Moderate activity |

### How do you convert pIC50 to IC50? ›

Simply stated, **pIC50 is the negative log of the IC50 value when converted to molar**. For example, an IC50 of 1 uM is 1 x10^-6 M and that's equal to a pIC50 of 6. An IC50 of a nanomolar compound is 1 x10^-9 M, which is a pIC50 of 9.

### What is IC50 value in cytotoxicity? ›

Most of the synthesized compounds (1b–1e and 2a–2e) exhibited cytotoxic activity against HeLa cell lines with IC50 value in the range of **40.65–95.55 µM**.

### What is IC50 in MTT assay? ›

The IC50 value (that is, **the concentration of drug which exhibited 50% cell viability for MCF-7 and MDA-MB-231 cells**) were 15, 15, 10 μM, and 100 nM, respectively, for AZA, SAM, SFN, and TSA, respectively. Source publication.

### How do you perform an IC50 experiment? ›

IC50 or cell viability experiment - YouTube

### Is higher or lower IC50 better? ›

The value of f_{∞} describes drug efficacy—the lower the f_{∞} the higher the beneficial effect (often denoted by E_{max}, maximal effect). The IC_{50} value is correlated with drug potency, i.e. the amount of drug necessary to produce the effect—**the lower the IC _{50} value the more potent the drug** [33].

### What does a high IC50 mean? ›

IC 50 is the dose of the drug required to kill 50%of cancer cells. The smaller the IC 50, more potent the drug is. **Drugs having large IC50 can be toxic and can have side effects**.

### Is IC50 concentration dependent? ›

In enzyme assays usually IC50 is independent of concentration. But **if you consider simple receptor ligand binding assay IC50 is dependent of receptor concentration**. So, selecting the appropriate receptor concentration is very important.

### Is IC50 and EC50 the same? ›

The concepts of IC50 and EC50 are fundamental to pharmacology. **The EC50 is the concentration of a drug that gives half-maximal response.** **The IC50 is the concentration of an inhibitor where the response (or binding) is reduced by half**.

### Why do we use IC50? ›

Half-maximal inhibitory concentration (IC50) is the most widely used and informative measure of a drug's efficacy. It **indicates how much drug is needed to inhibit a biological process by half, thus providing a measure of potency of an antagonist drug in pharmacological research**.

### How do you convert to nanomolar? ›

**1 Molar [M] = 1 000 000 000 Nanomolar [nM]** - Measurement calculator that can be used to convert Molar to Nanomolar, among others.

### How is IC50 cytotoxicity calculated? ›

We used the following sigmoidal dose-response formula to calculate the IC50 values: **Y=Low CI + (High CI-Low CI)/{1+10 ^ (Log IC50-X)}**, where 'Low CI' represents the minimum CI values, 'High CI' represents the maximum cell index values, Y is the cell index, and X is the log of concentration (M).

### How do you write IC50? ›

The change in IC_{50} due to competition is mathematically described by the Cheng–Prusoff relationship: **IC _{50}/K_{I} = 1 + [S]/K_{m}**, where [S] is the substrate concentration and K

_{m}is the Michaelis constant. The IC

_{50}is equal to K

_{I}for noncompetitive inhibition.

### How do you read MTT assay results? ›

The data is analyzed by plotting cell number versus absorbance, allowing quantitation of changes in cell proliferation. **The rate of tetrazolium reduction is proportional to the rate of cell proliferation**. Where PC is the Positive control and test is the sample being tested.

### What is IC50 cell viability? ›

The IC50 is defined as "**the concentration of an inhibitor where the response (or binding) is reduced by half**." So, if you are testing viability via an MTT assay, the dose of cytotoxic compound at which you achieve 50% viability will be the IC50.

### How do you calculate IC50 from inhibition? ›

**IC50 = (Conc.** **of tested agent × 50)/% inhibition**. So the IC50 value is a function of the %Inhibition value. Linear regression method is limited as it bases the IC50 on just concentration alone without considering the %inhibition at times especially when you have a control of same concentration with the extracts.

### How do you calculate the IC50 value of enzyme inhibition? ›

The change in IC_{50} due to competition is mathematically described by the Cheng–Prusoff relationship: **IC _{50}/K_{I} = 1 + [S]/K_{m}**, where [S] is the substrate concentration and K

_{m}is the Michaelis constant. The IC

_{50}is equal to K

_{I}for noncompetitive inhibition.

### How many concentrations does IC50 have? ›

The absolute EC50/IC50 should only be used if there are **at least two assay concentrations whose predicted response is less than 50% and two whose predicted response is greater than 50%**.

### What is IC50 experiment? ›

IC_{50} is **a quantitative measure that indicates how much of a particular inhibitory substance (e.g. drug) is needed to inhibit, in vitro, a given biological process or biological component by 50%**. The biological component could be an enzyme, cell, cell receptor or microorganism.

### How do you calculate IC50 value for antioxidant activity in Excel? ›

you can calculate the IC50 as follows: **IC50 = (0.5 - b)/a**. You replace (y) by 50 in the equation y = 1.3534 x + 0.58 and you deduce ( x) . Thank you all for answers.

### What is IC50 in antioxidant activity? ›

The IC50 value is a parameter widely used to measure the antioxidant activity of test samples. It is calculated as **the concentration of antioxidants needed to decrease the initial DPPH concentration by 50%** [23]. Thus, the lower IC50 value the higher antioxidant activity.

### What does EC50 stand for? ›

EC. _{50}. The parameter EC_{50} abbreviates for '**half maximal effective concentration**'. In a pharmacological context, this can be the concentration of a drug that is necessary to cause half of the maximum possible effect.

### Is IC50 and KM the same? ›

Besides, **IC50 is always larger than Ki**. By definition, IC50 is the “total” concentration of inhibitor needed to reach 50% inhibition; while Ki is the “free” concentration of inhibitor required to reach 50% enzyme saturation.

### What is the inhibitory constant? ›

Ki, the inhibitor constant

The inhibitor constant, Ki, is an indication of how potent an inhibitor is; it is **the concentration required to produce half maximum inhibition**. Plotting 1/v against concentration of inhibitor at each concentration of substrate (the Dixon plot) gives a family of intersecting lines.

### How do you calculate inhibitory concentration? ›

Abstract. The concentration of an inhibitor at which 50% inhibition occurs can be calculated for a linear regression curve of 1/v versus the concentration of the inhibitor by **dividing the intercept value by the slope of the curve**.

### What is potency of a drug? ›

Results: Potency is **an expression of the activity of a drug in terms of the concentration or amount of the drug required to produce a defined effect**, whereas clinical efficacy judges the therapeutic effectiveness of the drug in humans.

### What is EC50 and LC50? ›

EC_{50}(50% Effective Concentration)：Effective Concentration 50

**The Lethal Concentration 50 (LC50) is the concentration of a substance in water causing a death (50% of the tested population) to aquatic life**.

### What is EC50 vs ED50? ›

Therapeutic (Effective), Toxic, Lethal dose

* EC50 versus ED50: **EC50 is the dose required for an individual to experience 50% of the maximum effect.** **ED50 is the dose for 50% of the population to obtain the therapeutic effect**.

### What is the difference between potency and efficacy? ›

**Potency denotes the amount of drug needed to produce a given effect.** **Efficacy: Refers to the relative ability of a drug-receptor complex to produce a maximum functional response**.

### Why using pIC50 instead of IC50 will change your life? ›

Switching from IC50 to pIC50 **causes you to change how you think about your data and your experimental design**. It will encourage you to think logarithmically about your potency data and stop thinking about arithmetic scales.

### How much is Nanomolar? ›

1 Nanomolar is equal to **1E-09 Mole per Millimeter³**. 1 Nanomolar is 1000000000 times Smaller than 1 Mole per Millimeter³.

### What is Nanomolar concentration? ›

A nanomolar (nM) is **the decimal fraction of a molar**, which is the common non-SI unit of molar concentration. For example, a 2-molar (2 M) solution contains 2 moles of a certain substance in one liter of a liquid or gaseous mixture.

### How do you convert Nanomolar to picomolar? ›

The formula to convert Nanomolar to Picomolar is **1 Nanomolar = 1000 Picomolar**.

### How is IC50 value calculated in origin? ›

Curve Fitting: Origin: Dose Response Analysis - YouTube

### What is the difference between IC50 and CC50? ›

**IC50 is 50% inhibition conc.** **of sample dose and CC50 is cytotoxic conc.** **of sample on death of viable cells**.

### How do you calculate percentage inhibition in Excel? ›

The percentage of inhibition was calculated using the following formula: Percentage of inhibition: **(Control OD − (Sample OD/Control OD)) × 100**.

### How do you find the LC50 of a graph? ›

In order to determine the LC50, you first need to **figure out the concentrations of sediment, then graph them against the mortality**. Have a computer fit a best-fit line to the graph, then find where the line crosses the 50% mortality mark.

### How do you find the absolute IC50 of a prism? ›

From the data table, click Analyze, choose nonlinear regression, and choose the panel of equations: Dose-Response -- Special, X is log(concentration). Then choose "Absolute IC50, X is log(concentration)". You must enter a value for the parameter "Baseline" on the Constrain tab of the analysis parameters dialog.

### Why is DMSO used in MTT assay? ›

We have found that **DMSO is the best solvent for dissolving the formazan product**, especially where a significant amount of residual medium is left in the wells of the microtitre tray used for the assay.

### Can cell viability be more than 100? ›

**Yes indeed, you can get more than 100%**. Yes, In case of cellular proliferation we can get higher percentage of cellular viability using calculation of ratio between treated cell and non-treated/standard cell OD values in percentage.

### How do you calculate cell viability from cytotoxicity? ›

The percentage of cell viability is calculated using the following equation: **% Viability = A 450 − A 650 of test cells A 450 − A 650 of control cells × 100** .

### How do you calculate IC50 from inhibition? ›

**IC50 = (Conc.** **of tested agent × 50)/% inhibition**. So the IC50 value is a function of the %Inhibition value. Linear regression method is limited as it bases the IC50 on just concentration alone without considering the %inhibition at times especially when you have a control of same concentration with the extracts.

### How do you calculate the IC50 value of enzyme inhibition? ›

The change in IC_{50} due to competition is mathematically described by the Cheng–Prusoff relationship: **IC _{50}/K_{I} = 1 + [S]/K_{m}**, where [S] is the substrate concentration and K

_{m}is the Michaelis constant. The IC

_{50}is equal to K

_{I}for noncompetitive inhibition.

### How do you calculate IC50 value for antioxidant activity in Excel? ›

you can calculate the IC50 as follows: **IC50 = (0.5 - b)/a**. You replace (y) by 50 in the equation y = 1.3534 x + 0.58 and you deduce ( x) . Thank you all for answers.

### Is higher IC50 better? ›

The value of f_{∞} describes drug efficacy—the lower the f_{∞} the higher the beneficial effect (often denoted by E_{max}, maximal effect). The IC_{50} value is correlated with drug potency, i.e. the amount of drug necessary to produce the effect—**the lower the IC _{50} value the more potent the drug** [33].

### How is IC50 value calculated in origin? ›

Curve Fitting: Origin: Dose Response Analysis - YouTube

### How do you calculate lc50 in Excel? ›

Probit and LC50 calculation using MS Excel - YouTube

### Does IC50 change with enzyme concentration? ›

The short answer to your enzyme inhibitor IC50 question is **yes, the enzyme concentration does affect the measured IC50 of its inhibitor**, especially if the inhibitor is of the competitive inhibitor variety.

### Does IC50 depend on substrate concentration? ›

Many functional assays seek a total inhibitor concentration that reduces these activities by 50% (IC_{50}). However, **the IC _{50} value depends on concentrations of the enzyme (or target molecule), the inhibitor, and the substrate (or ligand)** along with other experimental conditions.

### Is IC50 and EC50 the same? ›

The concepts of IC50 and EC50 are fundamental to pharmacology. **The EC50 is the concentration of a drug that gives half-maximal response.** **The IC50 is the concentration of an inhibitor where the response (or binding) is reduced by half**.

### Is IC50 and KM the same? ›

Besides, **IC50 is always larger than Ki**. By definition, IC50 is the “total” concentration of inhibitor needed to reach 50% inhibition; while Ki is the “free” concentration of inhibitor required to reach 50% enzyme saturation.

### What is IC50 value in DPPH? ›

The IC50 value is **a parameter widely used to measure the antioxidant activity of test samples**. It is calculated as the concentration of antioxidants needed to decrease the initial DPPH concentration by 50% [23]. Thus, the lower IC50 value the higher antioxidant activity.

### What is IC50 in pharmacology? ›

**Half-maximal inhibitory concentration** (IC50) is the most widely used and informative measure of a drug's efficacy. It indicates how much drug is needed to inhibit a biological process by half, thus providing a measure of potency of an antagonist drug in pharmacological research.

### How do you calculate antioxidant activity? ›

**4.**

**Chemical Tests Determining Antioxidant Activity**

- The ORAC Test. The ORAC test measures the splitting ability of the radical chain reaction by antioxidants through monitoring the inhibition of the oxidation of the peroxyl radical. ...
- The HORAC Test. ...
- The TRAP Test. ...
- The TOSC Test.

### What does a high IC50 mean? ›

IC 50 is the dose of the drug required to kill 50%of cancer cells. The smaller the IC 50, more potent the drug is. **Drugs having large IC50 can be toxic and can have side effects**.

### What is IC50 IC80? ›

IC50 and IC80 are **common metrics used to establish clinical expectations of bnAb activity from experimental results in vitro and to identify bnAbs with high potential for advancement into clinical trials**. While useful, these parameters alone offer only a limited description of neutralization activity.

### What is IC50 of antibody? ›

The IC50 is defined as **the concentration of antigen that blocks 50% of antibody-phage binding to immobilized antigen**.