Protein purification assays

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

We have previously shown that a 209 amino acid region (aa288-497, asymmetric localization domain) of Insc is necessary and sufficient for apical cortical localization and for mitotic spindle orientation along the apical-basal axis (Tio et al 1999). In a yeast two-hybrid screen we identified Partner of Inscuteable (Pins), a novel 658aa protein with multiple repeats of the Tetratricopeptide (TPR) motif. Affinity purification experiments using embryonic extracts demonstrate that Pins complexes with Insc in vivo. In vitro protein interaction assays demonstrates that Pins interacts with the Insc asymmetric localization domain (see Yu et al 2000). 

Directed evolution relies on the analysis of large numbers of clones to enable the discovery of rare variants with unproved function. In order to analyze these large libraries, methods of screening or selection have been developed, many of which use specialized equipment or automation. These range from the use of multichannel pipettes, all the way up to robotics, depending on the level of investment [59]. Specialized robotic systems are available to perform tasks such as colony picking, cell culture, protein purification, and cell-based assays. 

Volume 200. Protein Phosphorylation (Part A Protein Kinases Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression)... 

In addition to MALDI-TOF and LC-MS/MS, SELDI-TOF-MS can also be used to determine expression profiling of various biological samples, such as serum or plasma for early detection of infection. Serum proteomic profiling assay, for instance, has been used to distinguish patients with acute SARS (severe acute respiratory syndrome) from patients with fever and influenza with 100% accuracy [16]. A major limitation of SELDI-TOF-MS, however, is that it cannot be used for direct amino acid sequence identification of the biomarker proteins, necessitating further sample fractionation and protein purification. 

All purification procedures require a method for quantifying or assaying the protein of interest in the presence of other proteins. Purification can be monitored by assaying specific activity. 

Recall from the previous discussion of protein purification that an important step is the development of a specific assay for the desired protein. The assay must be specific so that the desired protein can be detected both... 

During a protein purification procedure there are two measurements that need to be made, preferably for each fraction. Measurements of the amount of total protein and of the amount (usually bioactivity) of the desired protein both must be made. It is not possible to isolate a protein without a method of determining whether it is present an assay, either quantitative or at least semiquantitative, indicating which fraction contains the most of the desired protein is essential. 

Throughout the purification process we must have a convenient means of assaying for the desired protein, so we can know the extent to which it is being enriched relative to the other proteins in the starting material. In addition, a major concern in protein purification is stability. Once the protein is removed from its normal habitat, it becomes susceptible to a variety of denaturation and degradation reactions. Specific inhibitors are sometimes added to minimize attack by proteases on the desired protein. During purification it is usual to carry out all operations at 5°C or below. This temperature control minimizes protease degradation problems and decreases the chances of denaturation. 

Yeang, H.Y., Yusof, F. and Abdullah, L. (1998) Protein purification for the Lowry assay acid precipitation of proteins in the presence of sodium dodecyl sulfate and other biological detergents. Analytical Biochemistry 265, 381-384. 

Purification protocols should always aim for brevity, to minimize complexity and cost. The goal of the protein purification procedure, besides a pure protein, is a purification table listing all the operations undertaken, with results on overall yield, specific activity, and purification factor. An assay for both protein function and protein concentration is required at every step. 

Subtilisin BPN was prepared through a series of protein purification steps applied to the fermentation broth. These steps included ultrafiltration ethanol precipitation DEAE (diethyl-aminoethyl) Tris Acryl batch anionic exchange SP (sulfopropyl) Tris Acryl column cationic exchange and, concentration with an Amicon stirred cell. The enzyme purity was determined to be -951 via spectroscopic assays that measure the ratio of active enzyme to total protein. In addition, purity was verified via HPLC and SDS-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 

In order to monitor the progress of the purification of a protein it is necessary to have an assay for it. Depending on the protein, the assay may involve measuring the enzyme activity or ligand-binding properties, or may quantify the protein present using antibodies directed against it. 

In cases where it has proved impractical to assay for activity at each step, protein purification has been aided by tagging a fraction of the molecules with a photoaffinity reagent (e.g. the lactose carrier of Escherichia coli Newman et al., 1981 and the /J-adrenergic receptor of frog erythrocytes Shorr et al., 1982). 

Figure 1. Key steps in the development of protein impurity assays. The reference impurities may be obtained by a process specific purification of host cell proteins arising from a blank run or a production run. While the production run is a more accurate population of potential impurities, the product removal step involves significant technical difficulty.

One of the most important requirements for successful protein purification is the availability of an accurate, rapid, and quantitative assay that is specific for the protein of interest. If the protein has no known enzymatic activity, the amount of the protein present after each purification step must be analyzed by some general method such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, or an enzyme-linked immunosorbent assay (ELISA). If... 

Another example of on-line monitoring of enzyme activities was given by Kunnecke et al. [88], when a FIA-system was used for the determination of enzyme activities during protein purification by fast protein liquid chromatography (FPLC). Photometric assays for four different oxidases were established in a FIA-system extending the linear range by the so-called zone sampling method. The FIA-device was coupled to the FPLC unit behind a... 

Lor large scale protein purification the need to assay for target proteins and critical impurities is often essential. In practice, when a protein is purified for research purposes, it is too time consuming to identify and set up specific assays for harmful contaminants. A practical approach is to purify the protein to a certain level, and then perform SDS-PAGE after a storage period to check for protease cleavage. Suitable control experiments, included within assays for bio-activity, will help to indicate if impurities are interfering with results. 

Washed cell pastes were diluted with fresh buffer using a ratio of 3 ml of buffer for each 1 g of cell paste. A French Cell Press operated at 16,000 lb/in was used to disrupt cells. The crude enzyme preparation was then centrifuged at 10,444 X g for 30 min at 0°C, and the supernatant was retained as a cell-free extract (CFE). This extract was carried through a series of activation and purification steps and the methlonlnase activity was assayed after each step using a modification of the method of Tanaka t al. (52). Protein was assayed by the dye-binding method of Bradford (62), and enzyme solutions were dialyzed in tubing prepared by procedures described by Brewers at al. (63). 

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