Of protein solutions

Koenig S H and Brown R D 1990 Field-cycling relaxometry of protein solutions and tissue— implications for MRI Prog. Nucl. Magn. Reson. Spectrosc. 22 487-567... 

Ball V and Ramsden J J 1998 Buffer dependence of refractive Index Increments of protein solutions Biopolymers 46 489-92... 

Van den Berg, G.B. Hanemajer, J.H. and Smolders, C.A., "Ultrafiltration of Protein Solutions the Role of Protein Association in Rejection and Osmotic Pressure," Journal of Membrane Science, 31 (1987) 307-320. 

The shaking of protein solutions may lead to aggregation and precipitation as a result of several mechanisms, such as air oxidation, denaturation at the interface, adsorption to the vessel, or mechanical stress. These possibilities were systematically examined for solutions of human fibroblast interferon [50]. In this example, mechanical stress was identified as the causative factor in the inactivation. The proposed mechanism of inactivation by mechanical stress was through orientation of the asymmetrical protein in the... 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

Add 25 pi of the stock solution of either SPDP or LC-SPDP in DMSO to each ml of the protein to be modified. If sulfo-LC-SPDP is used, add 50 pi of the stock solution in water to each ml of protein solution. 

To release the pyridine-2-thione leaving group and form the free sulfhydryl, add DTT at a concentration of 0.5 mg DTT per mg of modified protein. A stock solution of DTT may be prepared to make it easier to add it to a small amount of protein solution. In this case, dissolve 20mg of DTT per ml of 0.1M sodium acetate, 0.1M NaCl, pH 4.5. Add 25 pi of this solution per mg of modified protein. Release of pyridine-2-thione can be followed by its characteristic absorbance at 343 nm (s = 8.08 X 103M 1cm 1). 

To deprotect the acetylated sulfhydryl group of SAMSA-modified proteins, add 100 pi of 0.5 M hydroxylamine hydrochloride in 50 mM sodium phosphate, 25 mM EDTA, pH 7.5, to each ml of protein solution. 

Dissolve cystamine (Aldrich) in the reaction buffer at a concentration of 2.25mg/ml (lOmM). Add an aliquot of this solution to the protein solution to be modified. Use about a 10- to 20-fold molar excess of cystamine over the amount of protein present. For a protein of MW 100,000 at a concentration of lOmg/ml, add 10pi of the stock cystamine solution to each ml of protein solution to obtain a 10-fold molar excess. 

Apply to the column 1.0ml of protein solution (dissolved in equilibration buffer-2) to be reduced. The inclusion of a denaturant in the solution deforms the protein structure so that inner disulfides are available to the immobilized reductant. Without the presence of guanidine or another deforming agent (i.e., urea, SDS, etc.), only partial reduction of the protein is possible. 

Add the solution prepared in step 2 to the protein solution to obtain at least a 10-fold molar excess of small molecule to protein. In the case of the peptide-protein immunogen conjugate, add the 500 pi of peptide solution to the 200 pi of protein solution. 

In a darkened lab, slowly add 50-100 pi of FITC solution to each ml of protein solution (at 2 mg/ml concentration). Gently mix the protein solution as the FITC is added. 

In subdued lighting conditions, add 25-50 pi of the fluorescein solution to each ml of protein solution while mixing. Alternatively, determine the exact molar quantity of protein present and add a 25-fold molar excess of fluorescein-5-maleimide solution. 

Reduce disulfides in the two protein samples by the addition of 2 pi of 50 mM TCEP (Thermo Fisher) to each 100 pi aliquot of protein solution. Cover and boil the samples for 10 minutes in a water bath to completely denature and reduce the proteins. Avoid the use of thiol-containing reductants, such as DTT, as these will react with the iodoacetyl group on the ICAT compound. 

Add 0.5 ml of protein solution to each ml of liposome suspension with stirring. 

Add 10 mL of colloidal gold to 0.2 mL of protein solution containing the minimal amount of protein needed to stabilize the gold plus 10% (as determined in step 3) (see Note 2). 

A fully automated system for performing detailed studies has been developed to improve the reproducibility and throughput (Fig. 12.2) [8]. It consists of two functional components a sample-deuteration device and a protein processing unit. The preparation operations (shown at the top of Fig. 12.2) are performed by two robotic arms equipped with low volume syringes and two temperature-controlled chambers, one held at 25 °C and the other held at 1 °C. To initiate the exchange experiment, a small amount of protein solution is mixed with a deuter-ated buffer and the mixture is then incubated for a programmed period of time in the temperature-controlled chamber. This on-exchanged sample is immediately transferred to the cold chamber where a quench solution is added to the mixture. 

Equation 3 Indicates that a semilog plot of flux against concentration should be a straight-line intercepting the horizontal axis at the gel concentration (C ). When the bulk-stream concentration (C ) equals the gel concentration (C ) there is no driving force for removal of solute from the membrlne. The gel layer increases in thickness until the flux is zero. Figure 10 provides experimental confirmation for a number of protein solutions and colloidal suspensions ( ). The Intercepts with the horizontal axis are reasonable values for the gel concentration. 

Add 1 pil of protein solution to the same well in the same way. The two (separate) 1 -p.1 drops join and become a 2-p.l drop. If the drops don t coalesce, mix them gently with the pipette tip. 

The influence of neutral salts as well as of acids and bases on the swelling of gelatine which we have seen can be attributed to an apparent change in the solvation of the gel fibrils and may be interpreted in the light of Donnan s theory of the effect of a non-diffusible ion on the osmotic pressure differences between the two phases, is likewise to be noted in the alteration of the viscosity and alcohol precipitation values of protein solutions. From the considerations already advanced there should exist two well-defined maxima in the viscosity and alcohol precipitation curves when these properties are plotted as functions of the Ph, the maxima coinciding with the points of maximum dissociation of the salts... 

To precipitate a protein, mix its solution either at RT or 4 °C with the required amount of saturated ammonium sulfate solution (consider the different concentration of (NH4)2S04 at RT and 4 °C). As an example, mix 6 vol. of protein solution with 4 vol. of saturated ammonium sulfate solution to get 40% saturation. 

Pipet 5-10 pi of Soln. A (4-7 MBq) into a 4-ml polystyrene or siliconized glass test tube. Then add 25 pi Soln. B, followed by 10 pi of protein solution (2-5 pg, dissolved in Soln. C), 10 pi Soln. D, and 100 pi Soln. E. Vortex after each addition. Fill up to 1 ml with Soln. G and count for total radioactivity. 

Pipet about 5-10 MBq (about 250 000-500 000 dpm) ofSoln. Ainto a 4-ml test tube. Carefully vaporize the solvent by a gentle stream of dry nitrogen. Add 10 pi of protein solution, containing 2-5 pg of protein in Soln. B, and agitate the tube in an ice bath for 30 min. Add 0.5 ml Soln. C and continue shaking at 0 °C. Fill up to 1 ml with Soln. D after an additional 5 min. Separate the labeled protein from the unreacted reagent as described in Protocol 6.4.1. 

The amount of surface adsorption of a number of proteins ranging in molecular mass from 6.5 to 670 kDa and isoelectric point (pi) from 4.3 to 10.5 to several commonly used container surfaces (glass vials either untreated, siliconized, sulfur treated or Purcoat treated plastic vials polyester + 0.3%, polyester 5x0, PP, and nylon). A 5-mL volume of protein solution was added to each vial, yielding a surface-to-volume ratio of 2.4cm2/mL. No correlation was found between the amount adsorbed and the molecular mass or isoelectric point, although glass surfaces appeared to bind more protein under the experimental conditions examined [156]. 

Size-exclusion chromatography can be used to analyze protein-protein interactions. Bloustine et al. (2003) presented a method to determine second virial coefficients (B2) of protein solutions from retention time measurements in size-exclusion... 

Kinsella (13, 14) summarized present thinking on foam formation of protein solutions. When an aqueous suspension of protein ingredient (for example, flour, concentrate, or isolate) is agitated by whipping or aeration processes, it will encapsulate air into droplets or bubbles that are surrounded by a liquid film. The film consists of denatured protein that lowers the interfacial tension between air and water, facilitating deformation of the liquid and expansion against its surface tension. 

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