Extinction coefficient calculation

  • Required for accurate protein quantification by UV absorbance
  • Compare batch-to-batch concentrations
  • Suitable for purified protein and antibody samples

Do you need the molar extinction coefficient of a drug substance protein?

It is important to know the protein’s extinction coefficient – regardless of you follow the ICH Q6B Guidelines or not. When you do, you can use spectrophotometry and UV-absorption to determine the protein concentration in laboratory samples.

The coefficient depends main...

Do you need the molar extinction coefficient of a drug substance protein?

It is important to know the protein’s extinction coefficient – regardless of you follow the ICH Q6B Guidelines or not. When you do, you can use spectrophotometry and UV-absorption to determine the protein concentration in laboratory samples.

The coefficient depends mainly on the actual protein/antibody sequence, but also on absorbing amino acids and the buffer composition. Fortunately, it is possible to determine it accurately.

Our standard procedure:

First we determine the exact quantity of protein/antibody by amino acid analysis. Then we prepare a dilution series of your protein and measure the UV absorbance at 280 nm at different protein concentrations. Finally, we calculate the molar extinction coefficient from the dilution series and the protein molecular weight.

You receive:

  • Determination of the molar extinction coefficient of your protein/antibody.
  • Dilution series with UV absorbance at 280 nm as function of concentration.
  • Quantitative amino acid analysis of your protein stock solution.
  • Calculation of protein molecular weight from sequence.
  • Analysis result in 2-3 weeks.

You are welcome to send us a request if you have specific analysis needs.

MoreLess details

The process:

  • 1 You provide us with purified protein, AA sequence and buffer
  • 2 We perform triplicate amino acid analysis for accurate quantification
  • 3 We determine the UV absorbance at 280 nm for a dilution series
  • 4 We calculate extinction coefficient for the protein in your buffer
  • 5 You receive results within 2 weeks

More information:

See an example of how results are presented

Get sample report Extinction-coefficient

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We would like to help you as much as possible with your project and therefore provide several kinds of customer support:

Technical details

Introduction to Extinction Coefficient calculation

When the concentration of a given protein is known, the concentration and molecular weight can be used to determine the molar extinction coefficient.

It is determined using the Beer-Lambert law: A = ε ∙ L ∙ cA is the absorbance at 280 nm, L is the light path (cm), c is protein concentration (g/L), and ε is the extinction coefficient (L g-1cm-1). When the light path = 1cm, ε = A/cε value corresponds to the slope of the line plotted between absorbance and concentration.

You then calculate  am (L mol-1cm-1) using the formula am = ε∙ MW, where MW is the molecular weight of the protein.

Extinction Coefficient calculation

 

Extinction Coefficient Diagram

Sample preparation

Sample requirements for Extinction Coefficient calculation

What we need from you to perform this analysis:

  • 1-3 mg purified protein/antibody.
  • The protein amino acid sequence.
  • The buffer to be used for measurement.
  • Concentration range to cover.

If you do not provide the buffer along with the sample we will use PBS as a standard buffer system.

Related services

You can combine this analysis with other analyses such as:

Meet the experts

For this type of analysis our experts include:

Do you need help?
Thanh Ha Nguyen

MSc in Biotechnology

Read more
Do you need help?
Per Aaslo

MSc in Molecular Biology & Environmental Biology

Read more
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