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# Protein Concentration Calculator

How do I calculate protein concentration?Understanding the parameters — absorbance, extinction coefficient, molecular weight, ...How to use the protein concentration calculator — ExampleTable of extinction coefficients and molecular masses for spectrophotometryFAQs

Welcome to the protein concentration calculator! 🧪

This calculator will determine the protein concentration of your stock or diluted sample. Simply insert your spectrophotometer data and choose your protein or amino acid! Jump right into the calculation or keep reading to:

• Learn the formula for protein concentration;
• Go through a step-by-step example calculation; and
• Get explanations for all parameters of the protein concentration formula, such as the extinction coefficient, pathlength, and molecular weight.

## How do I calculate protein concentration?

The formula for protein concentration from absorbance is derived from the Beer-Lambert Law, $A= \varepsilon\times b\times C$ (see our Beer Lambert Law calculator for more details), which determines the concentration of chemicals that absorb light. Modifying the Beer-Lambert Law, you receive the formula for protein concentration:

$C = \frac{A}{\varepsilon \times b} \times m\times n$

where:

• $C$Protein concentration;
• $A$Absorbance at λmax;
• $\varepsilon$Extinction coefficient;
• $b$Pathlength of the cuvette;
• $m$Molecular mass; and
• $n$Dilution factor.

## Understanding the parameters — absorbance, extinction coefficient, molecular weight, ...

To be able to calculate the protein concentration of a sample, we need to know the values of the five variables:

• Absorbance;
• Extinction coefficient;
• Pathlength;
• Molecular mass; and
• Dilution factor.

#### Absorbance 🌈

The absorbance or optical density (OD) can be measured with a spectrophotometer. The value tells us how much light a sample absorbs at a given wavelength. We want to measure at λmax where the substance's wavelength experiences the strong absorbance. For proteins, λmax is normally at 280 nm wavelegth.

#### Extinction coefficient 🔎

The extinction coefficient tells you how strong a substance absorbs or reflects light at a particular wavelength. It is specific for different substances and varies between proteins since it depends on their atomic and chemical structure. You will likely find the extinction coefficient in your protein or amino acid manual.

#### Pathlength 🔬

The pathlength is simply the inner diameter of the cuvette you use. Typically, this value equals 1 cm.

#### Molecular mass ⚛

It is the atomic mass of the substance divided by the amount of a substance, expressed in grams per mol (g/mol). Take a look at our molecular mass calculator or our specific protein molecular weight calculator to determine this value and find out more.

#### Dilution factor 🧪

You need to consider this parameter if you don't measure the absorbance of a stock sample but of a dilution. For example, if you have a 1:2 dilution, you would enter the multiplier 2 into the equation. If you need help with your dilution, our dilution factor calculator is ready to help you!

## How to use the protein concentration calculator — Example

Let's look at an example to find out how the protein concentration calculator works:

1. Choose your protein or amino acid from the options of the calculator's drop-down menu. Choosing your substance will automatically set the values for this protein's extinction coefficient and molecular mass. The calculator will show the values right under the Protein variable.

If you cannot find your substance in the menu, you can choose "Custom protein" to give the calculator your protein's specific extinction coefficient and molecular mass values. You can also find a table for the extinction coefficients and molecular masses of all available proteins of the calculator in this table.

Let's say we need to find out the concentration of an Immunoglobin G (IgG) sample, which has an extinction coefficient of 210,000 M⁻¹ cm⁻¹ and a molecular weight of 150,000 g/mol.

2. Insert the wavelength at which you measured the maximum absorption. We measured our sample at the standard 280 nm, so we enter this value into the calculator.

3. Enter the dilution factor. If you use a stock sample, type the value 1. If you are working, for example, with a 1:2 dilution, type in the value 2. In our example, we use a 1:10 dilution, so we type 10 into the according calculator field.

4. Verify the pathlength of the cuvette. The calculator uses a default value of 1 cm, but you can adjust it if necessary. We will use a standard cuvette with a pathlength of 1 cm, so we don't need to do anything here.

5. Let the calculator do the math! Using the protein concentration formula, the calculator will immediately show you the result:

$\quad\footnotesize \begin{split} C &= \frac{280\ \text{nm} \times 150,\!000\ \text{g/mol} \times 10}{210,\!000\ \text{M⁻¹ cm⁻¹} \times 1}\\[1em] &= 2000\ \rm mg/mL \end{split}$

## Table of extinction coefficients and molecular masses for spectrophotometry

Look at the following table to find the extinction coefficients and molecular masses of all the proteins and amino acids available in this calculator. The values are based on the research of and of von Hippel and Gill and are valid for measurements at absorbance of 280 nm.

Substance

molecular mass $m$ [g/mol]

extinction coefficient $\varepsilon$ [M⁻¹ cm⁻¹]

Aldose

38,994

34.380

APC – Allophycocyanin

105,000

700,000

ATP

507.2

15,400

BSA – Bovine Serum Albumin

66,463

43,824

Cysteine

121.16

120

IgG – Immunoglobin G

150,000

210,000

Insulin

5734

6335

Lysozyme

14,000

37,901

PE – Phycoerythrin

240,000

1,960,000

Phenylalanine

165.19

200

RNAse A

13,700

9440

Streptavidin

55,000

176,000

Tryptophan

204.23

5500

Tyrosine

181.19

1490

FAQs

### How to measure protein concentration?

Standard methods for measuring the protein concentration are measuring the UV absorbance with a spectrophotometer (normally at 280 nm) or by reacting the protein with dyes and/or metal ions (e.g., Bradford protein assay or BCA assays).

### How to calculate protein concentration from absorbance 280?

To calculate the protein concentration C at an absorbance A = 280 nm, you also need to know:

• The extinction coefficient ε, e.g., for Immunoglobin G 210,000 M⁻¹ cm⁻¹;
• The molecular mass m, e.g., 150,000 g/mol;
• The pathlength of the cuvette b, e.g. 1 cm; and
• The dilution factor n, e.g., 2.

Insert you values into the formula for protein concentration in mg/mL:

C = (A / (ε × b)) × m × n
C = (280 / (210,000 × 1)) × 150,000 × 2
C = 400 mg/mL

### How do I calculate protein concentration in µg mL?

To convert the protein concentration to µg/mL, you need to first calculate the protein concentration in mg/mL and then multiply the result by the conversion factor 1000.

If your protein concentration is, for example, 2000 mg/mL, you would calculate:

2000 mg/mL × 1000 = 2,000,000 µg/mL
= 2×10⁶ µg/mL

### How does Bradford protein assay work?

Protein concentration measurement with the colorimetric Bradford protein assay is based on the shift in absorbance maximum of the dye Coomassie Brilliant Blue G-250 from 465 to 595 nm following binding to denatured proteins in solution.

Under acidic conditions, the red form of the dye is converted into its blue form, binding to the protein being assayed. If there's no protein to bind, then the solution will remain brown.