Of concentrated protein solutions

As the next step, suitable sample conditions have to be found which maximize the protein s solubility and stability in order to prepare samples that are stable for several weeks, allowing the spectroscopist to collect all necessary spectra without having to prepare a new sample after each experiment. In particular, if aggregation has been detected, solution conditions have to be screened to produce a monodispersed protein solution. The most elegant way to screen many different solution conditions with a minimum amount of protein is the use of either the microdialysis button [9] or the microdrop screen [10, 11]. In these screens, small amounts (1-5 pL) of concentrated protein solutions are... 

Fig. 12.2 Diagram of a fully automated system for acquiring H/D exchange MS data starting with a stock solution of the nondeuterated protein. In this system [8], the liquid handler mixes a small amount of concentrated protein solution with a selected deuterated buffer and the mixture is incubated for a programmed period of time. The exchange reaction is conducted in a temperature-controlled chamber held at 25 °C. The mixture is then transferred to an acidic quench solution held at 1 °C. After quenching the exchange reaction, the entire sample is injected onto an LC-MS system...

Vilker, V.L., Colton, C.K., and Smith, K.A. The osmotic-pressure of concentrated protein solutions - effect of concentration and ph in sahne solutions of bovine serum-albumin. Journal of Colloid and Interface Science, 1981, 79, No. 2, p. 548-566. 

As a general rule, concentration polarization will be all the more important as membrane permeability and liquid viscosity will be higher and solute diffusivity will be lower. This is the recison why its effects, ordinarily negligible for nanofiltration, appear to be of major importance during ultrafiltration. Under gel polarization conditions, i.e. in those cases where reaches a maximum gel value Cg (as an example with ultrafiltration of concentrated protein solutions). 

Once a well-characterized, pure reference standard of known concentration is available, one must be aware of the fact that the actual preparation of the assay calibrators can also influence the PK standard curve. The physical treatment and handling of concentrated protein solutions can be very challenging. Some reference standards require specific handling techniques to prevent aggregation or denaturation of the protein. The effects of vortexing can be disastrous as can the process used to thaw frozen reference standard or calibrator solutions. Details such as the dilution pattern that is used in spiking the calibration solutions can contribute to assay bias and between-laboratory discrepancies. 

Limited enzyme treatment significantly reduced the viscosity of concentrated protein solutions and emulsification capacities were increased (5 ). The enzyme-treated proteins had slightly increased water absorption and foaming properties, but foam and emulsion stabilities were decreased (54). 

The micellar solvent system used in this assay has several advantages, in addition to rapid separation SDS is an effective-denaturing agent and, as such, can be used to stop reactions without the need for protein precipitation with trichloroacetic acid or heating. Since SDS also has a unique solubilizing power, it can be used for direct injection of concentrated protein solutions into the RPLC system, without time-consuming steps to remove protein precipitates or extraction of folate analogues. Therefore, the assay is simple, rapid, inexpensive, and applicable to crude hydrolase preparations. 

Food scientists working with biologic tissues usually encounter mixed proteins in the concentration range of 5-20%. The use of concentrated protein solutions maximizes protein-protein interactions and results in calorimetric data with a marked dependence on water content. 

Figure 18.4 The hanging-drop method of protein crystallization, (a) About 10 pi of a 10 mg/ml protein solution in a buffer with added precipitant—such as ammonium sulfate, at a concentration below that at which it causes the protein to precipitate—is put on a thin glass plate that is sealed upside down on the top of a small container. In the container there is about 1 ml of concentrated precipitant solution. Equilibrium between the drop and the container is slowly reached through vapor diffusion, the precipitant concentration in the drop is increased by loss of water to the reservoir, and once the saturation point is reached the protein slowly comes out of solution. If other conditions such as pH and temperature are right, protein crystals will occur in the drop, (b) Crystals of recombinant enzyme RuBisCo from Anacystis nidulans formed by the hanging-drop method. (Courtesy of Janet Newman, Uppsala, who produced these crystals.)...

Precipitation is another method of concentration that is used extensively for biopolymers such as proteins, polypeptides, etc. By increasing the salt concentration of a protein solution, some proteins can be precipitated. The sample is then centrifuged and the supematent liquid removed. The protein can be reconstituted in an appropriate solution, usually the one that will subsequently be used as the mobile phase. 

The rejection coefficient R) was calculated according to the following equation / = In (Cr/C )/ln (VJVr). Cr or Vr represent the protein concentration in the retentate or the volume of the retentate Co is the concentration of the protein in the solution before filtration 1 is the initial volume of the feed. The pH value of each protein solution was immediately measured after dissolving the proteins in distilled water. 

Dissolve the Traut s reagent (Thermo Fisher) in water at a concentration of 2mg/ml (makes a 14.5 mM stock solution). The solution should be used immediately. For the modification of IgG at a concentration of lOmg/ml using a 10-fold molar excess of Traut s reagent, add 45.8 pi of the stock solution to each ml of the protein solution. 

Add a quantity of adipic acid dihydrazide or carbohydrazide (Aldrich) to the protein solution to obtain at least a 10-fold molar excess over the amount of aldehyde functionality present. High molar ratios are necessary to avoid protein conjugation during the reaction process. If the concentration of aldehydes is unknown, the addition of 32mg adipic acid dihydrazide per ml of the protein solution to be modified should work well. 

Add an aliquot of the hydrazine-modified protein solution to the p-nitrobenzaldehyde solution and incubate at 37°C for 1 hour or at room temperature for 2 hours. To assure accuracy, determine the linear response range of the test by adding a series of different concentrations of the hydrazine-modified protein solution to the p-nitrobenzaldehyde buffer. This is done by preparing a set of serial dilutions of the protein solution and... 

Add a quantity of the protein solution to the QD solution with mixing to obtain the desired molar excess of protein over the concentration of nanoparticles. Using a 1- to 20-fold molar excess typically works well, but optimization should be done to determine the best ratio for a particular application. 

With mixing, add a quantity of the sulfo-NHS-biotin solution to the protein solution to obtain a 12- to 20-fold molar excess of biotinylation reagent over the quantity of protein present. For instance, for an immunoglobulin (MW 150,000) at a concentration of 10 mg/ml, 20 pi of a sulfo-NHS-biotin solution (containing 8 X 10-4 mmol) should be added per ml of antibody solution to obtain a 12-fold molar excess. For more dilute protein solutions (i.e., 1-2 mg/ml), increased amounts of biotinylation reagent may be required (i.e., 20-fold molar excess or more) to obtain similar incorporation yields as when using more concentrated protein solutions. 

The NHS ester end of NHS-LC-biotin reacts with amine groups in proteins and other molecules to form stable amide bond derivatives (Figure 11.4). Optimal reaction conditions are at a pH of 7-9, but the higher the pH the greater will be the hydrolysis rate of the ester. Avoid amine-containing buffers which will compete in the acylation reaction. NHS-LC-biotin is insoluble in aqueous reaction conditions and must be solubilized in organic solvent prior to the addition of a small quantity to a buffered reaction. Preparation of concentrated stock solutions may be done in DMF or DMSO. Nonaqueous reactions also may be done with this reagent for the modification of molecules insoluble in water. The molar ratio of NHS-LC-biotin to a... 

In the nltTa.filtTa.tion of a protein solution of concentration 0.01 kg/m3, analysis of data on gel growth rate and wall concentration Cw yields the second order relationship ... 

A 10.0 ml solution containing proteins is reacted with biuret solution and the absorbance is measured as 0.356. Another 10.0 ml sample of the same protein solution is mixed with 5.0 ml of a protein solution known to contain 1.6pgml-1 of protein. The mixture is reacted in the same way as the original unknown and the absorbance is found to be 0.562. What is the concentration of the protein in the sample ... 

Both sulfonyl chloride and isothiocyanate will hydrolyze in aqueous conditions therefore, the solutions should be made freshly for each labeling reaction. Absolute ethanol or dimethyl formamide (best grade available, stored in the presence of molecular sieve to remove water) should be used to dissolve the reagent. The hydrolysis reaction is more pronounced in dilute protein solution and can be minimized by using a more concentrated protein solution. Caution DMSO should not be used with sulfonyl chlorides, because it reacts with them. 

Raman spectra obtained on concentrated protein solutions (ca. 20 mM) are dominated by contributions from the repeating amide group of the polypeptide backbone and from the sldechalns of aromatic amino acids (6). Phenylalanine and tryptophan exhibit particularly Intense ring breathing modes at 1006 and 1014 cm, respectively. An additional strong peak due to tryptophan Is typically observed at 760 cm ... 

The ultrasound contrast agents are manufactured from nontoxic natural or synthetic biodegradable materials (e.g., lipids or proteins), and a small amount of an inert low-solubility non-reactive gas (e.g., perfluorocarbon). These components have been shown to be harmless to the patient unlike the tens of milliliters of concentrated viscous solutions of the widely used X-ray contrast agents (which may sometimes result in nephrotoxicity). 

Many other biological structures exhibit LC behavior. For instance, the concentrated protein solution that is extruded by a spider to generate silk is actually an LC phase. The precise ordering of molecules in silk is critical to its renowned strength. DNA and many polypeptides can also form LC phases. Since biological mesogens are usually chiral, chirality often plays a role in these phases. 

Chang TMS. Stablisation of enzymes by microencapsulation with a concentrated protein solution or by microencapsulation followed by cross-linking with glutaraldehyde. Biochem Biophys Res Commun 1971b 44 1531-1536. 

The smaller the height of the sample, the better is the resolution. But a compromise has to he made between concentration and volume, because highly concentrated protein solutions tend to form precipitates on gels. 

As a variant, activate the peptide separately first and couple then to the carrier Dissolve the peptide to 1 mg/ml in ddH20, add 10 mg EDAC hydrochloride per milligram of peptide, and adjust pH to 5.0. Incubate at RT for 5 min and correct the pH with diluted NaOH during this period. Then add the same volume of carrier protein solution. The amount of carrier protein should be in a ratio of 40 moles of COOH groups per mol peptide (ovalbumin 42.7 kD, 31 Asp, 48 Glu/Mole BSA 67.7 kD, 54 Asp, 97 Glu/Mole). Shake at RT for 4 h and stop the reaction by addition of 1/10 volume of 1 M sodium acetate buffer, pH 4.2. Free the sample from surplus reagents by gel filtration or dialysis and concentrate to about 1 ml by ultrafiltration. 

---