Use with proteins

Lueking et al. (1999) arrayed recombinant proteins on NC membranes and screened them with different antibodies. Joos and coworkers (2000) printed down autoantigens onto NC membranes and compared performance relative to silylated (aldehyde) and PLL glass slides. Protein arrays could be stored at room temperature for a month without significant loss in activity. Huang (2001) hand spotted down IgC species and antibodies directed toward various cytokines onto membranes. The properties of various commercial membranes were assessed in terms of absorption, background, and sensitivity levels based upon detection by enhanced chemiluminescence (ECL). 

Advantages — High binding capacity multiple hybridization cycles possible. 

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Porous silica packings do, however, sometimes suffer from adsorption between the sample and silanol groups on the silica surface. This interaction can interfere with the size exclusion experiment and yield erroneous information. In many cases, this problem is easily overcome by selecting mobile phases that eliminate these interactions. In addition, the surface of porous silica packings is routinely modified in order to reduce these undesirable interactions. Trimeth-ylsilane modified packing is typically used with synthetic polymers. Diol modified packing is typically used with proteins and peptides. 

If the fluorometric procedure is used, with protein precipitation, then bilirubin will not interfere with the hexokinase procedure. Even if protein precipitation is not resorted to, the interference from bilirubin does not become significant until one is dealing with an infant in severe jaundice. This can be seen in Table III. 

The main advantage of NMR spectroscopy is its use with proteins in solution. In consequence, rather than obtaining a single three-dimensional structure of the protein, the final result for an NMR structure is a set of more or less overlying structures which fulfill the criteria and constraints given particularly by the NOEs. Typically, flexibly oriented protein loops appear as largely diverging structures in this part of the protein. Likewise, two distinct local conformations of the protein are represented by two differentiated populations of NMR structures. Conformational dynamics are observable on different time scales. The rates of equilibration of two (or more) substructures can be calculated from analysis of the line shape of the resonances and from spin relaxation times Tj and T2, respectively. 

Conventional plastic microtiter plates have also been adopted for use with protein microarrays in the "array of arrays" format. Matson et al. (Oak Ridge... 

Although low expression levels limit the application of FRET methods, acceptor photobleaching FRET can be successfully used with proteins expressed at as low as 100-150 thousand per cell. 

The simple thermodynamic cycle must be expanded for use with proteins because there are so many ionizing groups with overlapping pA s. If <2D(pH) and <2N(pH) are the number of protons bound to the denatured state and the native state, respectively, then... 

Malathion [121-75-5], 0,0-dimethyl -(l,2-dicarbethoxy)ethyl phosphorodithioate (60) (bp 156—157°C at 93 Pa, d 1.23, vp 5.2 mPa at 30°C), is soluble in water to 145 mg/L. The rat LDBOs are 1375,1000 (oral) and 4000 (dermal). Malathion readily hydrolyzes in water above pH 7.0 and below pH 5.0. It is one of the most widely used general-purpose insecticides by virtue of its low mammalian toxicity and its good persistence, and is effective for the home garden, household, and against insects of public health importance including flies, mosquitoes, and lice. It is used with protein hydrolysate bait to control fruitflies (Tephritidae). 

Today, the two most common LC/MS interfaces are atmospheric pressure ionization interfaces, electrospray (ESI) and ion spray (ISI). Electrospray (Fig. 15.8) and its subtype, nanospray, are recommended for use with proteins and highly polar or ionized compounds. They are very soft ionization, concentration-dependent techniques that result in very little fragmentation and often produce multiply charged molecular ions. 

Fig. 7.5. The repressed transactivator system can be used with proteins that activate transcription independently [33], Since the protein being screened, AX (activating protein X), has the intrinsic ability to activate transcription of the URA3 reporter gene, expression of AX makes the...

The solubility of proteins and nucleic acids in aqueous solution depends on the solvation of the macromolecule by water this can be influenced by pH, ionic strength and temperature, and also by the addition of salts, or water-soluble organic solvents. We discuss below the various precipitation methods that have been used with proteins and nucleic adds, particularly with regard to concentration and fractionation procedures. 

The electrophoresis technique most often used with proteins is SDS-PAGE, i.e., polyacrylamide gel electrophoresis carried out in the presence of 0.1 % sodium do-decyl sulphate (See and Jackowski 1989). This technique, which was developed by Shapiro, Vinuela and Maizels, separates proteins, or more accurately protein subunits, exclusively on the basis of their molar mass. SDS is an anionic detergent that binds to proteins up to a level of about 1.4 g g 1 of protein, and in so doing it disrupts the quaternary, tertiary and, to a large extent, the secondary structure of the... 

Steuerle and HUle (1959) and Hille (1960) developed a method for the quantitative determination of the N-terminal residues normally present in wool. After treatment with l-fluoro-2,4-dinitrobenzene the wool is hydrolyzed and the ether-soluble DNP derivatives applied to a column of nylon 66 powder and developed with phosphate buffer at 60°C. The DNP derivatives of aspartic acid, glutamic acid, serine, threonine, glycine, alanine, and valine separate cleanly and can be readily determined in the eluates. Hence, it is well suited to the determination of N-terminal residues in normal animal fibers. In its present form, however, it is not suitable for general use with proteins or modified wool fibers as some DNP derivatives, such as those incorporating two DNP groups, are bound so strongly by the nylon that they cannot be eluted. 

In this chapter, we have reviewed some critical aspects of stoppers that are used for packaging of lyophilized biopharmaceuticals. We have listed the desirable attributes for lyophilization closures for use with protein formulations. We have discussed issues relating to preparation of stoppers. Two issues, siliconization and occluded water, have been dealt with in detail. Siliconization can be a cause of particulate generation and aggregation, as shown by our experience with recombinant tumor necrosis factor formulations. We have described methods of measurement of stopper-occluded moisture and discussed their relative merits and demerits. We further studied the kinetics of moisture removal from stoppers under selected conditions. Our approach to validation of moisture removal from stoppers has also been presented. Finally, we have discussed the usefulness of occluded moisture measurement for determination of moisture uptake by protein products under real... 

Procedure 1 pepsin, subtilisin, aminopeptidase M, prolidase This method may often be useful with proteins that can be maintained in a- largely denatured state at acid pH. The protein (usually about 10 mg per ml) may be denatured in solvents like 6 M guanidinium chloride or 8 M freshly deionised urea and dialyzed exhaustively against 5 % formic acid at room temperature at about pH 2. If cysteine or cystine residues are present, these should be reduced and alkylated ( 3.8.2) prior to enzymic hydrolysis. 

Sorbic acid is an antimicrobial preservative with antibacterial and antifungal properties used in pharmaceuticals, foods, enteral preparations, and cosmetics. Generally, it is used at concentrations of 0.05-0.2% in oral and topical pharmaceutical formulations, especially those containing nonionic surfactants. Sorbic acid is also used with proteins, enzymes, gelatin, and vegetable gums. It has been shown to be an effective preservative for promethazine hydrochloride solutions in a concentration of 1 g/L. ... 

Protein precipitation is often used as the initial sample-preparation scheme in the analysis of small drug molecules, since one universal procedure is followed for all compounds and method development is unnecessary. The speed of this technique presents a real time savings. The high resolving power of LC-MS/MS analytical methods accommodates this nonselective cleanup procedure. This mode of sample preparation is also inexpensive and does not chemically alter the analyte. Typical sample matrices that are used with protein-precipitation techniques are plasma, serum, tissue homogenates, and in vitro incubation mixtures. In general terms, a sensitivity of 1-10 ng/mL is often achieved from 50- 4L sample volumes with this technique with LC-MS/MS analysis. 

Siliceous earths 20-50 ml/hl 50-100 ml/hl Act on protective colloids in wines that are difficult to clarify. Used with protein fining agents, prevents overfining and facilitates settling of the lees... 

Methods are under development for analysis of protein from formalin-fixed tissues, although protein yield may be less than that from frozen tissue. Extraction of high-yield, quality protein from formalin-fixed tissue is generally difficult because of the extensive cross-links formed in formalin-fixed samples. Currently, the optimal tissue specimen for use with protein lysate microarrays is fresh tissue, immediately embedded in a cyroprotectant solution and frozen or snap-frozen tissue. 

Landianides can also be used with proteins which do not have intrinsic Ending sites. Reagents have been developed whidt can be coupled to proteins uid which chelate Ian-thanides. Because of dteir sensitivi to water, Ian thanide complexes designed as labels generally have most sites occupied by the ligand. 

The components of a sample are compelled to remain at their isoelectric point. This feature means that the technique should not be used with proteins which become insoluble or denatured at the isoelectric point. 

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