The Path to Protein Concentration Measurement

8th May 2018

Category: Bioanalytical

Tags: , ,

By: Stuart Wright - Scientist,

Protein Concentration Analysis

Quick and accurate measurement of protein concentration is very important within the biopharmaceutical industry. This must be carried out on a large number of samples prior to performing the various physicochemical analysis involved in protein characterization or protein structural analysis, since many assays depend on knowledge of the biotherapeutic protein concentration. However, acquiring measurements of a protein concentration in a sample can be a laborious task and inaccuracies can arise through sample handling (pipetting errors).

Spectrophotometry is a well-established technique for determining the concentration of a protein sample. It involves passing light at a specified wavelength through a sample and measuring the absorbance. The wavelength commonly used for this process is 280 nm, due to the strong absorbance of aromatic amino acids at this region of the UV spectrum. Hence the analysis is commonly referred to as ‘A280 Analysis’. The absorbance is related to the concentration using the Beer-Lambert Law:

A = εCL

Where A = absorbance, ε = extinction coefficient, C = concentration path, L = path length

 

Fixed vs. Variable Path Length Spectrophotometry

A fixed path length spectrophotometer requires samples to be carefully diluted to bring their concentration into the linear range of the instrument at the fixed path length. This is a time consuming process with any errors associated with the dilutions leading to errors in the final concentration calculated for the sample. Another downside of the technique is that it uses a portion of the sample which cannot be recovered.

A solution to the problems associated with fixed path length spectrophotometry is to use a variable path length instrument, such as the SoloVPE (C Technologies, Inc.) [1]. The SoloVPE alters another component of the Beer-Lambert Law, the path length, to perform the analysis.

In traditional fixed path length spectrophotometry a sample is placed inside a cuvette with a known path length. The light is then passed through the sample and the absorbance measured (Figure 1a). For the SoloVPE instrument, the sample is placed inside a cuvette, however, instead of the light passing through the sides of the cuvette, a fibrette, attached to a delivery fibre, is automatically lowered into the sample and light passed down through the sample (Figure 1b) [2]. This means that by lowering or raising the fibrette the path length can be altered. This brings the path length into the linear range of the instrument for a large range of concentrations without the need for dilutions.

BioOutsource Protein Concentration SoloVPE

Figure 1 a, b (respectively)

The SoloVPE generates a gradient from measurements of absorbance at different path lengths, within the linear range of the instrument (Figure 2). This gradient can be used to calculate the concentration due to it being linked to the Beer-Lambert Law as follows [1, 2]:

BioOutsource SoloVPE

Therefore:

BioOutsource SoloVPE

Where m = gradient of slope

BioOutsource SoloVPE graph

 Figure 2

Hence, using this approach, if the extinction coefficient is known then the concentration can be calculated from the gradient generated. Likewise, if the concentration is known then the extinction coefficient can be calculated.

Sartorius Stedim BioOutsource Protein Concentration Assay

Recent implementation of the SoloVPE instrument at Sartorius Stedim BioOutsource has led to development of a new protein concentration assay, qualified for samples in the range 0.1 mg/mL to 150 mg/mL (Figure 3). The assay is accurate and precise, helped by the fact that dilutions are generally not required. Its inherent speed means that a large number of samples can be analysed in a short amount of time, when compared to using a fixed path length spectrophotometer. Another benefit of using the SoloVPE instrument is that the sample is recoverable after analysis; useful when only a small amount of sample is available and a number of different tests are required.

BioOutsource SoloVPE concentration graph

  Figure 3

If you would like to find out more about our protein concentration assay using the SoloVPE instrument, click here to contact our expert scientists.

 


References

[1] www.solovpe.com – C Technologies, Inc.

[2] SoloVPE Quick Reference Guide (DOC025) – C Technologies, Inc.

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