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Products for Quantification

Quantification of Protein Extracts

Assays for Total Protein Concentration

Following protein extraction from cells or tissues, the protein concentration of the resulting solution must be determined for downstream analytical methods. An extracted protein preparation might be used in an enzyme activity assay, in which case an accurate assessment of the protein substrate is required for calculating reaction kinetics. More commonly, extracted protein mixtures are analyzed by SDS-PAGE, or they are fractionated on the Gelfree 8100 Fractionation System in preparation for analysis by LC-MS. In these instances, quantitative precision is essential to enable standardization of total protein loading, or to maximize total protein loading for preparative fractionation using the Gelfree system.

Choosing a Protein Quantification Method

Principal factors to consider when choosing a protein quantification method are the potential for bias in the amino acid compositions of the proteins to be quantified, and the reagent components present in the final extract solution.

Protein Extraction Bias

The accuracy of certain protein concentration assays can be adversely affected when the sample is biased towards proteins incorporating specific amino acid residues at high frequencies. For instance, the Bradford assay will produce artificially high results if the assayed solution contains many proteins high in arginine. Proteins high in cysteine produce disproportionately higher readings from the BCA assay. These limitations can be overcome by careful selection of internal protein quantification assay standards, assuming the investigator is aware of these differential sensitivities and of extraction biases affecting the relative ratios of certain amino acids in extracted protein solutions. However, when the objective is to measure the total protein concentration of a universal sample preparation containing an unbiased whole-proteome extraction, these considerations may be safely set aside.

Extraction Buffer Composition

Typical extraction buffer components such as chelating agents, detergents, and reducing agents are incompatible with one or more routine protein quantification methods. The Lowry assay cannot be used with proteins extracted into buffers containing effective concentrations of SDS or other detergents. Assays that depend upon metal-ion reduction to produce light-absorbing pigments are sensitive to chelating agents such as EDTA. And while the BCA assay as originally published is compatible with detergents commonly used in extraction buffers, it is not compatible with solubility-enhancing reducing agents such as DTT and ϐ-mercaptoethanol.

One way to accommodate these incompatibilities is to insert a protein precipitation step into the sample analysis workflow. Following precipitation, extracted proteins can be re-suspended and re-solubilized in a buffer solution compatible with the assay method of choice. This workaround requires extra work, time and materials, increases the risk that laboratory error will affect the experiment, reduces total protein yield, and introduces the possibility of sample degradation and unintentional biases in the resuspended sample. Most investigators would agree that it would be best, under most circumstances, to avoid the complications of this additional step by selecting an assay method that works correctly with buffers used to prepare the initial protein extract.

Quantifying Whole Proteome Extracts

Potential problems related to amino acid composition-dependent differential assay sensitivities are not a concern when the objective is to quantify unbiased, whole-proteome extractions such as those produced by the UPX UniversalProtein Extraction Kit or the YPX Yeast Protein Extraction Kit. The buffers included with these kits contain SDS, which is used to effect complete proteome solubilization. Therefore the detergent-compatible and readily scalable BCA assay is a preliminary candidate protein quantification assay for the whole-proteome preparations produced by these kits. Maintaining the solubility of the high quantity and diversity of proteins in UPX and YPX kits, however, requires the presence of reducing agents, and are present in their buffer solutions.

While the original BCA assay is not compatible with the presence of reducing agents, it has since been discovered that thiol group alkylation of the sample prior to the addition of color-producing assay reagents restores the efficacy of the BCA assay.

Proteoquant Proteome Quantification Assay Kit