Extinction coefficient calculation

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

In need of molar extinction coefficient of a drug substance protein?

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

The protein / antibody extinction coefficient depends mainly on the actual sequenc...

In need of molar extinction coefficient of a drug substance protein?

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

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

Our standard extinction coefficient calculation procedure consists of:

First, we determine the exact quantity of protein/antibody by our amino acid analysis service. 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:

  • Accurate calculation of the molar extinction coefficient of your protein/antibody.
  • Dilution series with UV absorbance at 280 nm as a function of concentration.
  • Quantitative amino acid analysis of your protein stock solution.
  • Calculation of molecular protein weight from a sequence.
  • You receive a written report.

You are welcome to send us a request with 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 10-20 work days

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We offer customized solutions, contact us to discuss your project.

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More information

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 protein 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 lawA = 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 the extinction coefficient am (L mol-1cm-1) using the formula am = ε∙ MW, where MW is the molecular weight of the protein.

Extinction Coefficient calculation

 

Antibody 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.

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

Sample Purity
We can analyze most samples that contain proteins and peptides.
Avoid high sucrose, urea (> 2 M) or salt concentrations.
Do not use buffer that contain glycerol and high amounts of primary amines, e.g. TRIS or glycine. Because it may interfere with the ninhydrin reaction and thus provide false quantification data of specific amino acids.
Avoid fingerprints and dust in the sample. These contain large amounts of human proteins that will obscure the results.

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

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Do you need help?
Line Kofoed

PhD in Health and Medical Sciences

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