Lidstrom:Choosing a protein concentration quantification method

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General Considerations

From ISBN 0-19-963142-5

• Each method depends to varying degree on the amino acid composition of the protein you are measuring, and usually involves comparion with a protein standard.
  • The mismatch between the protein standards' composition and your protein's composition is a source of error.
• The only way to measure protein concentration directly is to weigh the protein in question after complete removal of water and contaminants, which is rarely possible or desirable.
• Highly purified proteins in aqueous solution can be estimated more easily than crude homogenates or membrane-bound proteins where many additional components in the mixture may interfere. ISBN 0-19-963142-5 pg 327

Major methods available

Spectrophotometer

• Absorbance at 280 nm gives a quick approximate estimation of protein in non-turbid solutions, which is often all that is required. ISBN 0-19-963142-5
  • Advantage" non-destructive.
• A280 varies from protein to protein:
  • A280 for a 1% protein solution varies from 0 for some parvalbumins to 26.5 for lysozyme ISBN 0-19-963142-5 pg 327 though most fall in the range of 4-15.
  • A 1% BSA solution has A280 = 6.6.

Background info: ISBN 0-19-963142-5 pg 327

• Absorption at 280nm is due almost entirely to the aromatic amino acids: tryptophan and tyrosine.
• At 260 nm phenylalanine absorbs, and histidine, methionine, cystiene, and cystine absorb between 225 and 240 nm. At wavelengths less than 225 nm the peptide bond itself is the principal absorbing group, but the above amino acids also contribute.
• There are a number of reasons why these shorter wavelengths are not the most frequently used. At 192nm dissolved oxygen absorbs strongly, and thus measurements would have to be carried out in the absence of oxygen. 192 nm is below the working range of many spectrophotometers, since it requires a particularly good light source and stray radiation can be a problem. Wavelengths nearer 225 nm are used since, although the peptide bond absorbs less strongly, the interference by oxygen is limited.
• In a typical protein solution used in enzymatic studies, the main interfering substances likely to affect absorbance measurements are nucleic acids, nucleotides such as ATP or NAD(P), haem-containing compounds such as cytochromes, reagents containing sulphydryl groups such as 2-mercaptoethanol, dithiothreitol, glutathione or cystiene, and buffers.
• In general, the shorter the wavelength the larger the range of interfering substances. It is perhaps this reason that although A280 is less sensitive than some, and is dependent on the tyrosine and trypotphan content, it is one of the most used.
• The absorbance spectrum of tyrosine is pH dependent, but at 280nm both the protonated and unprotonated forms have similar absorption coefficients and so measurements do not have to be made at a particular pH.
• Some spectrophotometers used to measure column effluents use 206nm, which gives much greater sensitivity provided there is no interference.

Others

• Since the 1950s when Lowry et al. (8) modified the method originally devised by Wu (9) and Folin and Coicalteu (10), the Lowry method has become the most widely used for protein estimation. However, during the 1980s dye-binding assays based on Coomassie Blue (11,12) have become more popular because of their simplicity and because of certain shortcomings in the Lowry method. One of the oldest procedures, the Biuret test (13), is still used; it is quite satisfactory apart from the disadvantage of being relatively insensitive.
• Lowry depends to a significant extent on the aromatic amino acid content
  • The color yield for a protein like gelatin is about 1/4 the color that of trypsin. ISBN 0-19-963142-5 pg 327

Who uses what as of 2013

• Mila uses the spectrophotometer. She thinks hers is based on A260 and A230 (260 and 230 nm absorbances)
• Ceci/Amanda/Frances chose BCA, using a pierce brand kit.

Compatibility with your lysis solution

protein assay compatibility table from Pierce

Resources

• Thermo Scientific: Overview of Protein Assays Methods
• Thermo Scientific: Protein Assay Selection Guide
• Thermo Scientific: Protein Assay Technical Handbook