Protein, proteins sampling

Biotinylation of Target Protein. Protein samples are biotinylated essentially as described by the manufacturer (Pierce, Milwaukee, WI). Briefly, EZ-Link Sulfo-NHS-LC-Biotin is employed to attach a biotin molecule via a linker to primary amines on the surface of the target protein. The molar ratio of biotin linker to protein is adjusted so that each target protein molecule carries approximately one biotin. Overbiotinylation of the target protein is discouraged as this may alter the structure or function of the protein. 

For deterrnination of tryptophan, 4 M methanesulfonic acid hydrolysis is employed (18). For cystine, the protein is reduced with 2-mercaptoethanol, the resultant cysteine residue is carboxymethylated with iodoacetic acid, and then the protein sample is hydroly2ed. Also, a one-pot method with mercaptoethanesulfonic acid has been developed for tryptophan and cystine (19). 

Because protein samples are actually ampholytes, when samples are loaded onto the gel and a current is appHed, the compounds migrate through the gel until they come to their isoelectric point where they reach a steady state. This technique measures an intrinsic physicochemical parameter of the protein, the pi, and therefore does not depend on the mode of sample appHcation. The highest sample load of any electrophoretic technique may be used, however, sample load affects the final position of a component band if the load is extremely high, ie, high enough to titrate the gradient ampholytes or distort the local electric field. 

S mg), dimer (peak I) and monomer (peak 2), ovalbumin (S mg) (peak 3), and cytochrome c (3 mg) (peak 4) was loaded onto a Fractogel EMD BioSEC column (600 X 16 mm) with a bed height of 600 mm. PBS (pH 7.2) was used as the eluent at a flow rate of I ml/min the sample volume was O.S ml. (B) The same protein sample as in A was injected onto a column of identical dimensions packed with unmodified Fractogel HW 6S. Without the tentacle modification the base matrix displays only a poor resolution of the test mixture. 

Although the viscosity of the sample solution may affect the resolution, for practical reasons highly concentrated protein samples will give the best separations in the case of SEC with respect to the process economy. Although the actual loading capacity depends on the separation problem and on the... 

FIGURE 7.14 A Fractogel EMD BioSEC Superformance column (600-16) was loaded with 500 /il of BSA, ovalbumin, and cytochrome c (5/5/3 mg/ml) at I ml/min. The test covered 100 individual runs with the standard proteins as samples. The buffer system used was 20 m/VI sodium dihydrogen phosphate, 300 m/VI NaCI, pH 7.2. After each individual run the column was rinsed with I /VI NaOH (60 min with I /VI NaOH at 2 ml/min). No significant change in retention times and resolution was observed after 100 cycles. 

FIGURE 5.5 (a) The hydroxy amino acids serine and threonine are slowly destroyed during the course of protein hydrolysis for amino acid composition analysis. Extrapolation of the data back to time zero allows an accurate estimation of the amonnt of these amino acids originally present in the protein sample, (b) Peptide bonds involving hydrophobic amino acid residues snch as valine and isolencine resist hydrolysis by HCl. With time, these amino acids are released and their free concentrations approach a limiting value that can be approximated with reliability. 

Fig. 10. HPLC of proteins (commercial samples) on the /V-butyl polyacrylamide coated silica gel column. Sample 20 pi of 5-15 mg/ml protein solution in buffer A. Buffer A 10% methanol, 0.2 mol/1 ammonium acetate, pH 4.5. Buffer B methanol. Gradient 50-min linear, 0-100% B. Flow rate 0.8 ml/min. Peaks (/) — lysozym, (2,3) — insulin, (4,5) — myoglobin [57]...

Screening of compound libraries with medium throughput is only possible if the spectra can be recorded in a short period of time, and if one measurement gives simultaneously a number of answers. In practice, several ligands are usually measured in one protein sample, depending on the problem [5]. Mostly 10-20 ligands are combined to multiplexes, which need to be deconvoluted if a positive answer, as shown in Fig. 1 is obtained. The hits obtained maybe analyzed automatically or by hand through manual inspection of the spectra. 

This comparative study pointed out molecular close packing as a key parameter responsible for the thermal stability of proteins in films. In the case of BR, this close packing is reached due to the nature of the sample, while LB organization seems to be a more general procedure, for the same goal can be reached for practically any type of protein sample. The last statement was even confirmed by the comparison of the thermal behavior of extracted separated BR in self-assembled and LB films. It was found that BR in LB films is more stable for this kind of sample. The results will be reported in detail elsewhere. 

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Determination of neutral carbohydrate Total neutral carbohydrate in protein samples was estimated by the phenol/sulphuric acid method of Dubois [13] using mannose as standard. 

Fig. 9.40 NIS spectra of oxidized (filled circle) and reduced rubredoxin mutant Rm 2-4 (filled triangle) from Pyrococcus abyssi obtained at 25 K. The protein samples have been prepared with Fe concentrations of about 10 mM. Theoretically calculated NIS spectra based on DFT calculations (B3LYP/CEP-3IG) of 9, 21 and 49 atoms are shown below. The dotted lines represent calculated NIS spectra for the oxidized Fe S4 center and the dashed lines for the reduced Fe°S4 center. (Taken from [103])...

Because the pi of a protein is based on its amino acid sequence, this technique has good resolving power. The resolution can be adjusted further by changing the range of the pH gradient. The use of immobilized pH gradient (IPG) strips has enabled reproducible micropreparative fractionation of protein samples, which is not consistently possible when ampholytes are used in the first dimension (Gorg et al., 2000). 

A number of affinity-based or chromatography methods have been used to prefractionate protein samples for 2D electrophoresis. For example, proteins of low abundance can be enriched from crude lysates by affinity-... 

In recent years, a class of methods has been developed for molecular dynamics simulations to be performed with an external pH parameter, like temperature or pressure [18, 43, 44, 70], These methods treat the solution as an infinite proton bath, and are thus referred to as constant pH molecular dynamics (PHMD). In PHMD, conformational dynamics of a protein is sampled simultaneously with the protonation states as a function of pH. As a result, protein dielectric response to the... 

Ion exchange chromatography is another means to remove detergents and chaotropes from protein samples. This is one rarely mentioned, but well understood benefit of using ion exchange chromatography as a first dimension separation step in a two-dimensional LC experiment. 

---