|  Figure 4. The choice among available protein assays is usually based on the compatibility of the protein assay method with the samples. These data serve as a general guideline for evaluating response differences among protein samples. Comparison of point-to-point and linear-fit standard curves. Each protein in a sample responds uniquely in a given protein assay. The BCA assay has a lot of advantages. Therefore, successful use of protein assays involves selecting the method that is most compatible with the samples to be analyzed, choosing an appropriate assay standard, and understanding and controlling the particular assumptions and limitations that remain. Each method has its advantages and disadvantages. Most chemicals do not interfere with the assay. Selection of appropriate protein assay method for a paper microfluidics platform. Amino acids containing aromatic side chains (i.e., tyrosine, tryptophan and phenylalanine) exhibit strong UV-light absorption. NIH Peptide digest concentrations were determined using the Thermo Scientific Pierce Quantitative Colorimetric Peptide Assay and the Pierce Quantitative Fluorometric Peptide Assay Kits according to instructions. Clipboard, Search History, and several other advanced features are temporarily unavailable. No one reagent can be considered to be the ideal or best protein assay method. The preferred method of dealing with these types of interfering substances is to simply remove them. Instead, each protein assay detects one or several different particular amino acids with greater sensitivity than others. For example, tissues and cells are usually lysed with buffers containing surfactants (detergents), biocides (antimicrobial agents) and protease inhibitors. Disadvantages 1. For those samples that contain detergents, copper-based protein assays such as the Pierce Rapid Gold BCA assay are the better choice as they are not inhibited by low to moderate amounts of detergent. 2020 Jul 6;13(1):325. doi: 10.1186/s13104-020-05167-2. Because different pipetting steps are involved, replicates are necessary if one wishes to calculate statistics (e.g., standard deviation, coefficient of variation) to account for random error. Depending on the source material that the procedures involved before performing the protein assay, the sample will contain a variety of non-protein components. Data Brief. For this experiment, you will use a known solution of bovine immunoglobulin (1mg/ml IgG.) Get the latest research from NIH: https://www.nih.gov/coronavirus. Compatibility with the sample type, components, and workflows, Availability of the spectrophotometer or fluorometer needed to measure the output of the assay. Depending on the accuracy required and the amount and purity of the protein available, different methods are appropriate for determining protein concentration. The mechanism of protein molecule binding with the stain is described. doi: 10.1016/j.plabm.2020.e00166. Similar to protein assay methods, various options are available for determining peptide concentration. Each method has its particular advantages and disadvantages. Advantages/Disadvantages of Bradford Assay. The assay is separated into three main parts: preparation of the Diluted Albumin (BSA) Standards, preparation of the bicinchoninic acid (BCA) working reagent, and quantification of proteins (using either test tube or microplate procedure).. Because no one reagent can be considered the ideal or best protein assay method for all circumstances, most researchers have more than one type of protein assay available in their laboratories. cell lysates), Compatibility with most surfactants (detergents), Less protein–protein variation than the Coomassie dye–based assays, Incompatibility with substances that reduce copper, Incompatibility with common reducing agents such as DTT, Compatible with most salts, solvents, buffers, thiols, reducing substances, and metal-chelating agents, Incompatibility with surfactants (detergents), High protein–protein variation when compared to copper-based assays, Excellent sensitivity, requiring less protein sample for quantitation, Timing is not a critical factor, so the assays can be adapted for automated handling in high-throughput applications, Compatibility with the sample type and components, Protein-to-protein uniformity (see below), Speed and convenience for the number of samples to be tested, Availability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay. Advantages and disadvantages Advantages. DTT) and detergents (i.e. Pract Lab Med. For example, when the sample to be assayed is a purified antibody, bovine gamma globulin (BGG, protein #5) will be a more accurate standard than bovine serum albumin (BSA, protein #1). Analytical Biochemistry. Both BCA and micro-BCA assays are frequently used. (1985) On the chemical basis of the Lowry protein determination. Protocol. Again, the old dialysate is discarded and replaced with 1,000 mL of 100 mM PBS, with a pH of 7.6.