an_DL_045_Comparison_of_Test_Methods_for_Protein_Concentration
Application Note 487
Comparison of Test Methods for Protein Concentration
March 2011
Application Note
Comparison of Test Methods for Protein Concentration
1
Katie Slater B.S., 1Charles L. Crespi, Ph.D., 2Kristy Chatt, B.S.
1
Corning Life Sciences – Tewksbury, MA; 2BD Biosciences – Bedford, MA
Contents
1 Introduction 1 Methods
2 Results
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Introduction
An extracellular matrix (ECM) is a complex mixture containing glycoproteins, collagens, and proteoglycans which forms the structural framework that stabilizes tissues and provides mechanical support for cell attachment. The ECM exerts influences on cell behavior, and therefore, ECM reagents are widely used to study cell adherence, spreading, differentiation, migration, etc. One type of ECM, reconstituted basement membrane, is made up of sheets of ECMs (laminin, collagen IV, HSPG [perlecan], and entactin) that can cause the determination of protein concentration to be more difficult.
There are multiple suppliers of reconstituted basement membranes that use various methods of testing to determine protein concentration. Corning has consistently used the Folin-Lowry Protein Assay, an often-cited and standard method for protein determination. Other suppliers may use the Bicinchoninic Acid (BCA) assay method. These methods differ in sensitivity based on amino acid composition. The BCA method most readily detects cysteine/cystine, tryptophan, and tyrosine. The Lowry method is not as sensitive to proline residues. Reconstituted basement membrane, which can be rich in proline, may give higher readings with the BCA assay relative to the Lowry assay. Therefore, the same material assayed by two different methods may give different quantitative results even if the same reference protein is used.
Methods
We performed the following study of protein concentrations on reconstituted basement membranes. Corning ? Matrigel ? basement membrane matrix (Cat. No. 354234), Corning Matrigel basement membrane matrix, growth factor-reduced [GFR] (Cat. No. 354230), and comparable basement
membrane matrix products from another supplier were compared for protein concentration using the Lowry Method. Results were also compared to reported protein concentrations listed on the supplier specification sheets. In addition, 34 samples made up of a mixture of competitor and Corning Matrigel basement membrane matrix products (including both standard and GFR formulations) were assayed with both the Lowry and BCA methods.
BCA was measured using Pierce BCA Protein Determination kit, QCP-171. Lowry was
measured with Folin Phenol Reagent, QCP-028.
Application Note 487
Comparison of Test Methods for Protein Concentration
March 2011
Results and Discussion
Supplier A which uses the BCA method, reported protein concentrations on their certificates of analysis (C of A) that were 39% higher for standard product and 46% higher for GFR product than the concentration measured using the Lowry Method (as shown in Figure 1).
Figure 2. Comparison of Test Results from Lowry vs. BCA Method. The 34 samples tested by the Lowry and BCA methods show that the BCA method averages 20% (2.4 mg/ml) higher than the Lowry method when measuring the protein concentrations. When comparing between suppliers, one should be aware of the quantitative difference between BCA and Lowry assays for basement membrane matrices.
These differences may impact performance of assays or applications where basement membrane matrix is a key reagent.
Figure 1.
Comparison of Reported Protein Concentrations using Lowry Method
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