Comparison to Other Quantitation Methods

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Compare Direct Detect® IR-based protein quantitation with other methods traditionally used to measure total protein. These methods include Amino Acid Analysis, UV absorbance at 280 nm, and colorimetric assays such as BCA and Bradford assays.

• Comparison with Amino Acid Analysis
• Comparison with BCA and Bradford Assays
• Comparison with UV 280

Direct Detect® FTIR Protein Quantitation Matches Results Of Amino Acid Analysis

Amino acid analysis delivers possibly the most accurate protein quantitation; however, it is expensive and slow if samples are sent to a third party for analysis. If amino acid analysis is performed in-house, it requires time-consuming sample manipulation and specialized equipment. The Direct Detect® spectrometer provides protein quantitation in agreement with amino acid analysis (AAA), as shown*.

The Direct Detect® spectrometer provides faster protein quantitation and requires minimal amounts of often precious samples. Thanks to its new membrane technology for sample handling and presentation, the Direct Detect® method presents a more convenient alternative to amino acid analysis. The table below outlines the common features as well as distinguishing features of both methods.

Sample Compatibility with Direct Detect® Spectrometer vs. Amino Acid Analysis

Sample Characteristics	Amino Acid Analysis	Direct Detect® spectrometer
Buffer components containing “N-H” bond. Examples: Glycine Tris	Buffer exchange required: YES YES	Buffer exchange required: YES NO for concentrations < 0.2 M
Buffer components containing “C=O” bond	No limitations	Depending on concentration buffer exchange might be required
Contains Glycerol	Buffer exchange required (Causes artifacts and reduces some amino acid yields)	Buffer exchange required. (Does not dry well)
Contains Urea	Buffer exchange required. (Evolves to ammonia under hydrolysis conditions)	Buffer exchange required. (Strong absorbance in analysis region)
Contains DMSO	Does not dry well; Oxidizes Met, Trp, Tyr	Does not dry well; No limitations when dried
Contains Detergents	Cause artifacts and reduce some amino acid yields	Possible interference from detergents containing “N-H” bond (like CHAPS)
Amount requirements: Volume Mass Concentration	Per single analysis: Typically 10 – 100 µL Typically 1 to 10 µg Typically at 0.2 – 0.5 mg/mL	Per single analysis: 2 µL 0.5 µg From 0.25 mg/mL up
Associated error	5-30% on analysis of lysates 5-50% for single protein when sequence is not known	5-20% operator-related error associated with inaccurate pipetting

* For complete data and experimental details, download application note

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Comparison with BCA and Bradford Assays

Protein Quantitation Using Colorimetric Assays Can Be Inaccurate.

The most common colorimetric assays for protein quantitation involve protein-copper chelation (BCA and Lowry assays) and dye-binding based detection (Bradford and “660” assays). While these assays are easy to use, disadvantages include the large variation in the binding efficiency to different proteins, reproducibility, and sensitivity to sample contaminants.

Intrinsic protein characteristics that can affect concentration estimates in colorimetric assays include amino acid content, post-translational modifications, and protein secondary and tertiary structure. In one study, colorimetric assays gave results up to 60% different from values derived from amino acid analysis.

Bradford Assay: Factors Causing Its Inaccuracy

The Bradford assay relies on binding of Coomassie® Brilliant Blue G250 to basic amino acids, particularly arginine, and its absorbance shifts from 465 nm to ~595 nm upon binding. As a result, Bradford assay response depends on the number of basic amino acid residues in the protein. If the protein being assayed does not have a similar proportion of basic residues to the protein used for the standard curve, accuracy will be compromised.

Also, there may be a large variation in assay response between different preparations of the Bradford reagent. It has been shown that the absorbance maximum of the dye-protein complex varies between 595 nm and 620 nm, depending on the dye source. Bradford assay response is affected by detergents (such as those used to solubilize membrane proteins). Finally, the Bradford assay is nonlinear at the higher end of the recommended protein concentration range, making data analysis challenging and error-prone.

BCA Assay: Factors Causing Its Inaccuracy

The BCA assay involves a two-step chemical reaction. The first step requires protein binding to Cu²⁺, which is reduced to Cu¹⁺ by cysteine, tyrosine, tryptophan and peptide bonds. In the second step, BCA chelates Cu¹⁺ to form a purple complex that absorbs light at 565 nm.

Because it is so sensitive to the amino acid composition of the protein, the BCA assay, like the Bradford assay, requires a standard curve for each experiment, in which the protein standard has comparable amino acid composition to the protein being measured. For maximum sensitivity, the BCA assay requires heating, and assay signal can change depending on the length of incubation. The BCA assay is compatible with samples containing up to 5% ionic detergents; however, phospholipids, chelating agents, reducing agents, and certain nonionic, oxidizing detergents can affect the assay signal.

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IR-based Protein Quantitation Compared to Using Absorbance at 280 nm (A280)

The UV-Vis-based method of protein quantitation relies on absorbance at 280 nm by a protein’s aromatic amino acids, predominantly tryptophan with minor contributions from tyrosine. (Phenylalanine, although aromatic, does not contribute to absorbance at 280 nm).

Therefore, protein extinction coefficients can vary widely (greater than two-fold difference between extinction coefficients of albumin and immunoglobulin G). Those proteins that do not contain tryptophan, such as Protein A, are very difficult to quantify accurately based on 280 nm absorbance.

Also, secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum. In contrast, FTIR quantitation detects amide bonds, which are present in all proteins. Although protein structure influences the exact wavenumber of the Amide I band maximum, the intensity of this band can be used to accurately assess concentration.

What this means is that, as long as your protein is in a non-interfering buffer (see “Buffer Compatibility” section), you can use proteins like BSA as more universal standard curve, without being biased by the effects of protein sequence on absorption. In other words, standard curves made using these different proteins will have the same slope.

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