Concentration of Diluted Protein Solutions

Column Si. Size-exclusion chromatography columns are generally the largest column on a process scale. Separation is based strictly on diffusion rates of the molecules inside the gel particles. No proteins or other solutes are adsorbed or otherwise retained owing to adsorption, thus, significant dilution of the sample of volume can occur, particularly for small sample volumes. The volumetric capacity of this type of chromatography is determined by the concentration of the proteins for a given volume of the feed placed on the column. 

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... 

Prepare the protein to be modified in a non-amine-containing buffer at a slightly basic pH (i.e., avoid Tris or imidazole). The use of 0.1 M sodium phosphate, 0.15M NaCl, pH 7.2 works well for NHS ester reactions. The concentration of the protein in the reaction buffer may vary from pg/ml to mg/ml, but highly dilute solutions will result in less efficient modification yields. A protein concentration from 1 to 10 mg/ml works well in this reaction. 

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. 

Dissolve the molecules to be conjugated in 0.1M sodium phosphate, pH 7.2 (for aqueous reactions) or in DMSO, DMAC, or methylene chloride (for organic reactions). If proteins are to be conjugated, a concentration of l-10mg/ml in buffer will work well in this protocol. For more dilute protein solutions, greater quantities of the bis-NHS-PEG compound may have to be added than recommended here to obtain similar levels of crosslinking. 

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. 

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. 

The adsorption experiments were carried out by quantifying each of proteins adsorbed on the material from mono-component protein solutions, from four-component protein solutions, and from plasma and diluted plasma. Adsorption profiles of protein were largely different, depending on the aforementioned experimental conditions. For instance, the behavior of any particular protein from diluted plasma varied in response to the extent of plasma dilution. Cooper s results are illustrated in Fig. 3, on fibrinogen adsorption onto five polymer surfaces. It is seen that the adsorption profiles are different one another, being influenced by the different nature of the polymer surfaces. The surface concentrations of adsorbed protein are mostly time-dependent, and maxima in the adsorption profiles were observed. This is interpreted in terms of replacement of adsorbed fibrinogen molecules by other proteins later in time (Vroman effect). Corresponding profiles were also presented for FN and VN. 

Protein solution (protein, antibody) Depending on the estimated strength of binding, 10 to 0.1 p,g/mL corresponding to a dilution of 1 100 to 1 10,000 for a concentration of stock solution of 1.0 mg/mL (see Note 2). For a lower estimated affinity a higher concentration of the protein should be used. For protein mixtures (blood, plasma, cell extracts, etc.) estimation of the content of the target protein is necessary. 

A stock solution of rabbit Cytochrome c was made up in water at a concentration of 4.3 mg/ml. The concentration of the stock solution was determined by quantitative amino acid analysis of a 5pJ aliquot that was hydrolyzed and analyzed as previously described (4). The stock solution was diluted 5-fold with 0. IM ammonium bicarbonate or 0. IM sodium borate (both at pH 8.2) and TPCK-Trypsin in lOmM HCl was added to give a 1 20 enzyme protein solution (w/w). Digestion was carried out at 37°C for 24 hours after which the enzymatic activity was terminated by heating at 100°C for 5 min. 

Hydroxyapatite, when added to solutions of BSA in sufficient amounts, reduced the extinction of the protein solution at 280 nm to background, indicating total adsorption of protein. A similar experiment using diluted plasma gave no reduction in the extinction at 280 nm. It has been found, therefore, that hydroxyapatite cannot be used in the presence of significant concentrations of plasma or serum. Figure 4 shows the effect... 

Another approach to circumvent poor CE CLOD is to undertake offline sample pretreatment and analyte concentration. This should be avoided, if possible, particularly for dilute protein solutions since losses to exposed surfaces (e.g., walls of microcentrifuge tubes, pipette tips, solid extraction phases, etc.) can be substantial. Furthermore, excessive handing of a concentrated solution of protein(s) can lead to denaturation, aggregation, precipitation, and, ultimately, poor analyte recovery. 

An analysis of the cosolvent concentration dependence of the osmotic second virial coefficient (OSVC) in water—protein—cosolvent mixtures is developed. The Kirkwood—Buff fluctuation theory for ternary mixtures is used as the main theoretical tool. On its basis, the OSVC is expressed in terms of the thermodynamic properties of infinitely dilute (with respect to the protein) water—protein—cosolvent mixtures. These properties can be divided into two groups (1) those of infinitely dilute protein solutions (such as the partial molar volume of a protein at infinite dilution and the derivatives of the protein activity coefficient with respect to the protein and water molar fractions) and (2) those of the protein-free water—cosolvent mixture (such as its concentrations, the isothermal compressibility, the partial molar volumes, and the derivative of the water activity coefficient with respect to the water molar fraction). Expressions are derived for the OSVC of ideal mixtures and for a mixture in which only the binary mixed solvent is ideal. The latter expression contains three contributions (1) one due to the protein—solvent interactions which is connected to the preferential binding parameter, (2) another one due to protein/protein interactions (B p ), and (3) a third one representing an ideal mixture contribution The cosolvent composition dependencies of these three contributions... 

For our THz transmission measurements, we used solutions of thioredoxin in distilled water obtained from Sigma. The sample preparation technique and experimental procedure has been described in Ref Samples of diluted water solutions with the concentrations between 0.1 mg/ml and 3 mg/ml of proteins were measured. Several samples were made from each solution. Samples are in the form of a cell assembled from two polycarbonate films and a spacer (12 pm thick) placed between them, with 10 pi solution inside the spacer. Each sample was measured several times and the resulting spectra were calculated against the background of similar cells made from pure deionized water between two PC films. 

Using this relationship, the dissociation rate constant, k , was calculated as 3.91 X 10 min with a resultant half time of 177.3 minutes. In order to measure the rate constants for trimer formation, dissociation experiments were performed by rapid dilution of an equilibrated protein solution at association conditions (33 gM protein and 2.0 M GuHCl) to monomer conditions (3.3 mM protein, 2.0 M GuHCl). The diluted protein solution was analyzed by HPLC size exclusion to follow the dissociation over time (Figure 6). TTie distribution shifts to the monomer and dimer after ten minutes and the dimer slowly dissociates to form the monomer. The rate constant for trimer dissociation, k/, was calculated as 0.316 min (ti/2 = 2.19 minutes) from the rate of decrease in trimer concentration. Again, the relationship between the rate constants and the equilibrium constant was utilized. 

Bakerbond Application Note Bi-001 Extraction and Concentration of BSA Protein from Dilute aqueous Solution (See Suggested Reading, Chapter 1). 

Fig. 1. The vapor diffusion method for growing protein crystals. Water equilibrates from the more dilute protein solution in the drop into the more concentrated solution at the bottom of the beaker. This concentrates the solution slowly in the hanging drop.

---