Protein concentration, assays

Fortunately, protein concentration methods are relatively simple (low-tech) and inexpensive. The simplest assays require only a spectrophotometer calibrated for wavelength and absorbance accuracy, basic laboratory supplies, and good pipetting techniques. Protein concentration assays are quite sensitive, especially given the typical detection limits required for most biopharmaceuticals. 

This list of applications for protein concentration assays and the different requirements for each application reveal that no one method is superior to another. Each protein assay is chosen for a particular application based on its strengths in meeting specific requirements outweighing its weaknesses. Choosing the most suitable protein assay for the application is based on consideration of the following relevant questions and issues. 

BGA protein concentration assay reagents (other protein concentration assays such as Bradford can be used instead). Bovine serum albumin can be used for the standard. 

Perform a BCA protein concentration assay to determine protein concentration of the cell lysate see Note 12). 

Make sure to remove all media, especially those that contain fetal bovine serum (FBS) as proteins in FBS may interfere with the protein concentration assay and downstream protease concentration calculations. 

Any sufficiently sensitive protein concentration assay (e.g., Bradford) can be used to determine the protein concentration of the lysate. If the protein concentration of the lysate is less than 2 pg/pL, we recommend repeating the protein extraction using either more cells or less lysis buffer or concentrating the protein lysate see Note 7). A protein concentration of 2 pg/ pL in each 20 pL sample will provide sufficient protein for two Western blots if loading 20 pg per well. 

FIGURE 9. The effect of protein concentration of purified E. coli Met(0)-peptide reductase on the reduction of /V-acetylMet(O). Each incubation contained 5 x 10 4m /V-acetyl[3H]Met(O). The conditions and assay are described in the legend to Figure 8. Reproduced by permission of Academic Press from Brot and coworkers112. 

Limitations encountered in routine use include (a) the use of radioactive materials, (b) limited sensitivity in the presence of high protein concentrations, (c) long assay time of up to 5 days,... 

In current practice the fluorescence assay is often followed by the use of hybridization techniques when more selectivity is required. We have for instance used the fluorescence techniques to obtain data on the nucleic acid content of malaria vaccine proteins produced in Escherichia coli. The rapid turnaround time of the fluorescence assay is particularly useful during the early stages of purification to determine the optimal process conditions. After the final process has been arrived at and a variety of methods used to assess the nucleic acid content (including the hybridization techniques), the fluorescence method can be developed for routine quality-control purposes. In certain cases, particularly at high protein concentrations, the dye may bind to the protein with... 

Assays. Protein concentrations were measured by the method of Bradford (18) and the various contractile protein ATPase activities by tRe method of Martin and Doty (19). Gel electrophoresis was carried out by the method of Ames (20) on 1.5 ran polyacrylamide slabs using the discontinuous SDS buffer system of Laemnli (21). Dried gels were scanned at 550 nm for densiometry measurements. 

The specifications and standardization include raw materials, preparation method of the standard solution, concentration of proteins, and the main band on SDS-PAGE. The outline of the procedure for preparation of the calibrators is shovm in Eig. 4.2. Table 4.5 shows the raw materials and the preparation method of the initial extract. To prepare the calibrators, the raw materials are extracted by the standard solution containing SDS and mercaptoethanol. The initial extract is prepared by centrifugation and filtration of the extract. The diluted extract is then prepared by 10-fold dilution of the initial extract with phosphate-buffered saline (PBS pH 7.4). The protein concentration of the diluted extract is assayed using the 2-D Quant kit (Amersham Bio Sciences). The standard solution is then... 

In principle, the use of addressable, pooled GST fusion proteins could be used to identify proteins associated with any biochemical activity, assuming that the fusion protein is soluble, folded and functional. The method has the additional advantage that, once the GST fusion clones are constructed, it is a rapid technique. The authors state that only two weeks are required to purify the 64 pools and the assays can be accomplished in a day (Martzen et al., 1999). In addition, the method is sensitive because only 96 recombinant proteins are assayed at one time in contrast to the use of cell lysates where thousands of proteins are present. This leads to a much higher concentration of each protein, which greatly facilitates detection of a biochemical activity (Martzen et al., 1999). 

Figure 3. Conversion of DBT in vitro by lysate prepared from IGTS8 cells with assay protein concentration of 5 mg/ml. The data represents concentrations of DBT (circles), HPBS (diamonds), and HBP (squares). The data fitting represents a consecutive reaction model with kx = 0.2/min, and k2 = 0.05/min. Figure extracted from Ref [53].

Calculate the protein concentration in the final preparation using its absorbance at 280nm or a colorimetric method, such as the Coomassie assay. (Note The presence of hydrazine or hydrazide groups on the protein will interfere with the BCA assay for total protein concentration.)... 

For quantitative analysis of protein concentration the colorimetric Bradford-assay [147] is most commonly used. Here another Coomassie dye, Brilliant Blue G-250, binds in acidic solutions to basic and aromatic side chains of proteins. Binding is detected via a shift in the absorption maximum of the dye from 465 nm to 595 nm. Mostly calibration is performed with standard proteins like bovine serum albumin (BSA). Due to the varying contents of basic and aromatic side chains in proteins, systematic errors in the quantification of proteins may occur. 

In this method the keyhole limpet haemoglobin conjugate was prepared as follows Keyhole limpet haemocyanin (KLH, Calbiochem, La Jolia, CA) and bovine serum albumin (BSA, BDH Chemicals) were coupled to the adduct (2), derived from 6-bromohexanoic acid and monoquat (3), via a carbodiimide reaction, as reported previously by Niewola et al. [184], The resulting conjugates contained 662mol of Paraquat per mole of KLH and 15mol of Paraquat per mole of 6-bromohexanoic acid. The amount of Paraquat bound to the protein was determined by spectrophotometric dithionite assay for Paraquat and the protein concentration was established by a standard Lowry test. 

If analytical methods are at the heart of biopharmaceutical development and manufacturing, then protein concentration methods are the workhorse assays. A time and motion study of the discovery, development, and manufacture of a protein-based product would probably confirm the most frequently performed assay to be protein concentration. In the 1940s Oliver H. Lowry developed the Lowry method while attempting to detect miniscule amounts of substances in blood. In 1951 his method was published in the Journal of Biological Chemistry. In 1996 the Institute for Scientific Information (ISI) reported that this article had been cited almost a quarter of a million times, making it the most cited research article in history. This statistic reveals the ubiquity of protein measurement assays and the resilience of an assay developed over 60 years ago. The Lowry method remains one of the most popular colorimetric protein assays in biopharmaceutical development, although many alternative assays now exist. 

As described in the following chapter, there are many biopharmaceutical applications of protein assays. Assigning the protein concentration for the drug substance, drug product, or in-process sample is often the first task for subsequent analytical procedures because assays for purity, potency, or identity require that the protein concentration be known. Hence it is typical for several different methods to be employed under the umbrella of protein concentration measurement, depending on the requirements of speed, selectivity, or throughput. The protein concentration is valuable as a stand-alone measurement for QC and stability of a protein. However, protein concentration methods provide no valuable... 

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