Journal Article

Evaluating the efficacy of protein quantification methods on membrane proteins

Löptien, Jana, Vesting, Sidney, Mohammadi, Shabnam, Dobler, Susanne

2024

Overview

Protein quantification is an important tool for a wide range of biological applications. The most common methods include the Lowry, bicinchoninic acid (BCA) and Coomassie Bradford assays. Despite their wide applicability, the mechanisms of action imply that these methods may not be ideal for large transmembrane proteins due to the proteins’ integration in the plasma membrane. Here, we investigate this problem by assessing the efficacy and applicability of these three common protein quantification methods on a candidate transmembrane protein: Na, K-ATPase (NKA). We compared these methods with an ELISA, which we newly developed and describe here for the quantification of NKA. The use of a relative standard curve allows this ELISA to be easily adapted to other proteins and across the animal kingdom. Our results revealed that the three conventional methods significantly overestimate the concentration of NKA compared with the ELISA. This is due to the samples containing a heterogeneous mix of proteins, including a significant amount of non-target proteins. Further, by applying the protein concentrations determined by the different methods to in vitro assays, we found that variation in the resulting data was consistently low when the assay reactions were prepared based on concentrations determined from the ELISA.

• Publisher: The Royal Society,The Royal Society Publishing
• Subject: Amino acids / Animals / Antibodies / Antibodies - immunology / Cell Membrane - chemistry / Cell Membrane - metabolism / Chemistry Techniques, Analytical - methods / Chemistry Techniques, Analytical - standards / Coloring Agents - analysis / E coli / ELISA / Enzyme Assays - methods / Enzyme Assays - standards / Enzyme-linked immunosorbent assay / Enzyme-Linked Immunosorbent Assay - methods / Enzyme-Linked Immunosorbent Assay - standards / Freeze Drying / Genes / Limit of Detection / Membrane Proteins - analysis / Membrane Proteins - metabolism / Methods / Methods And Techniques / Na+/K+-exchanging ATPase / Na,K-ATPase / Peptides / Plasmids / Protein Quantification / Proteins / Reference Standards / Rosaniline Dyes / Sf9 Cells / Sodium-Potassium-Exchanging ATPase - analysis / Sodium-Potassium-Exchanging ATPase - metabolism / Staining and Labeling - methods / Staining and Labeling - standards / Transmembrane Proteins / Vertebrates