Sodium dodecyl sulfate serum albumin

Recently, novel polymethine carbonyl-dyes based on coumarin moiety and their boron difluoride complexes 9a-d and lOa-d [34—36] were evaluated as fluorescent dyes for the detection of native proteins using bovine serum albumin (BSA) as a model protein, and as probes for the nonspecific detection of proteins using a BSA/ sodium dodecyl sulfate (SDS) mixture [37]. Optical properties of these compounds in the absence and presence of BSA, as well as in SDS and BSA/SDS mixture, were measured in Tris-HCl buffer (pH 8.0) (Table 1). 

Figure 2.11. The dependence of the position of the fluorescence spectrum maximum on excitation wavelength for tryptophan in a model medium (glycerol) at different temperatures (a) and singletryptophan proteins (b). 1, Whiting parvalbumin, pH 6.S in the presence of Ca2+ ions 2, ribonuclease Th pH 6.5 3, ribonuclease C2, pH 6.5 4, human serum albumin, pH 7.0, +10"4 M sodium dodecyl sulfate 5, human serum albumin, pH 3.2 6, melittin, pH 7.5, +0.15 M NaCl 7, protease inhibitor IT-AJ from Actinomyces janthinus, pH 2.9 8, human serum albumin, pH 7.0 9, -casein, pH 7.5 10, protease inhibitor IT-AJ, pH 7.0 11, basic myelin protein, pH 7.0 12, melittin in water. The dashed line is the absorption spectrum of tryptophan.

High temperatures can break native S-S bonds and form new S-S bonds which can lock the protein into a denatured eonfiguration [89]. Low pH, sodium dodecyl sulfate. Tween 80, chaotropie salts, and exogenous proteins have been used to protect proteins from thermal inaetivation [90]. Ethylene glycol at 30-50% was used to protect the antiviral activity of P-interferon preparations [91]. Human serum albumin was used in recombinant human interferon-Psei-n which resulted in increased thermal stability [62]. Water-soluble polysaeeharides sueh as dextrans and amylose [92], as well as point-specific (site-directed) mutagenesis [93] have also been used to increase thermal stability of therapeutie proteins and peptides. 

JP Ou, STH Chang, WSB Yeung. Separation of bovine serum albumin and its monoclonal antibody from their immunocomplexes by sodium dodecyl sulfate-capillary gel electrophoresis and its application in capillary electrophoresis-based immunoassay. J Chromatogr B 731 389—394, 1999. 

The protein used was crystalline bovine serum albumin (BSA) obtained from Armour Laboratories. Sodium dodecyl sulfate (SDS) was synthesized from pure dodecyl alcohol and chlorosulfonic acid, recrystallized, and washed with ethyl ether. Sulfuric acid was reagent grade purchased from the J. T. Baker Chemical Co. and used directly. Reagent grade potassium hydroxide purchased from the Mallinkrodt Co. was purified by foaming a concentrated solution, removing the foam, and using the solution directly. 

Blots are prehybridized to block all nonspecific binding (which causes background). To do this, one first wets the nitrocellulose in 3 X SSC, then in 50% formamide, 3X SSC, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA (pH 8.0), 10 mM Tris (pH 7.5), 10X Denhardt s solution [1X Denhardt s is 0.02% Ficoll, 0.02% bovine serum albumin (BSA), and 0.02% polyvinylpyrrolidone 360], 0.05% sodium pyrophosphate, and 100 /ig/ml denatured DNA. Denatured DNA is sheared herring or salmon sperm DNA that has been boiled for 10 min before addition to the prehybridization mix. Blots are sealed within plastic bags, placed between two glass plates to give uniform distribution of prehybridization solution over the blot, and incubated in a water bath at 37° for at least 1 hr. 

Moriyama Y, Kawasaka Y, Takeda K. Protective effect of small amounts of sodium dodecyl sulfate on the helical structure of bovine serum albumin in thermal denaturation. J Colloid Interface Sci 2003 257(1) 41 6. 

Dimyristoylphosphatidylethanolamine (DMPE), biotinyl-N-hydroxysuccinimide ester (BNHS), triethylamine, myoglobin, bovine serum albumin (BSA), lysozyme, guanidinium chloride, dimethylamino-cinnamaldehyde, acrylamide, ammonium persulfate, sodium dodecyl sulfate (SDS), and molybdenum blue reagent were all obtained from Sigma Chemicals and used without further purification as received. The compounds N, N, N, N -tetramethylethylenediamine (TEMED), N, N -methylene-bis-acrylamide, and Coomassie Brilliant Blue R-250 were from BioRad Laboratories. Avidin was obtained from Vector Laboratories with a quoted activity of 14 units/mg. All solvents were from Fisher Scientific. Water was passed through a Barnstead Nanopure system. 

Protein interactions have been investigated by ITP (D6). It has, for example, been possible to determine the number of sodium dodecyl sulfate (SDS) molecules bound to bovine serum albumin (H14, H15) and to investigate the binding of drugs such as indomethacin to human serum albumin (H15). This method could be of value in pharmacokinetic studies. 

Figure 1 Relationships of S with interfacial tension and emulsifying activity of proteins. I, bovine serum albumin 2, /3-lactoglobulin 3. trypsin 4, ovalbumin 5, conalbuntin 6, lysozyme 7, K-casein 8, 9, I0, II, and 12, denatured ovalbumin by heating at 85°C for l, 2, 3, 4, and 5 min respectively 13, 14, 15, 16. 17, and 18. denatured lysozyme by heating at 85"C for l, 2, 3, 4, 5, and 6 min respectively 19, 20, 21, 22, and 23, ovalbumin bound with 0.2, 0.3, 1.7, 5.7, and 7.9 mol of sodium dodecyl sulfate/mol of protein respectively 24, 25, 26, 27, and 28, ovalbumin bound with 0.3, 0.9, 3.1,4.8, and 8.2 mol of linoleate/mol of protein respectively. Interfacial tension measured at corn oil/0.20c protein interface with a Fisher Surface Tensiontat Model 21. Emulsifying activity index calculated from the absorbance at 500 nm of the supernatant after centrifuging blended mixtures of 2 ml of corn oil and 6 ml of 0.5% protein in 0.01 M phosphate buffer, pH 7.4 S initial slope of fluorescence intensity (FI) vs. percent protein plot. 10 /al of 3.6 mM m-parinaric acid solution was added to 2 ml of 0.002 to 0.1% protein in 0.01 M phosphate buffer, pH 7.4, containing 0.002% SDS. FI was measured at 420 nm by exciting at 325 nm. (From Ref. 2. Reprinted by permission.)...

The purification used hydroxylapatite (HTP) from Biorad Inc., protamine sulfate, bovine serum albumin (BSA), and polylysine (Type VI) from Sigma Chemical Company, and epoxy-activated Sepharose from Pharmacia Inc. Sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing (IEF) were performed using chemicals and equipment from Biorad Inc. 

The specifications and standardization includes the raw materials, the preparation method for the standard solution, the concentration of protein, and the main band on SDS-PAGE. Table 15.1 shows the raw materials and the preparation method used for the initial extract. The preparation procedure for calibrators is as follows. The raw materials are extracted by a solution containing sodium dodecyl sulfate (SDS) and mercaptethanol. The initial extract is prepared by centrifuging and filtration of the extract. The diluted extract was then prepared by the 10-fold dilution of the initial extract with phosphate-buffered saline (PBS) (pH 7.4). The protein concentration of the diluted extract is assayed using the 2-D Quant kit (GE Healthcare). The standard solution is then prepared by a two fold dilution with PBS (pH 7.4) containing 0.2% bovine serum albumin (BSA). The calibrator included in each company kit is prepared by dilution of the standards (concentrated standard solution) to 50 ng/mL, with each company kit s original buffer containing the carrier protein. 

Nagata et al. reported the use of a polyethylene glycol-coated poly(methyl methacrylate) (PMMA) microchip for microchip gel electrophoresis separations. Trypsin inhibitor, bovine serum albumin (BSA) and 3-galactosidase (labeled with Alexa Ruor 488) were electrokinetically loaded for 60 s. The sodium dodecyl sulfate (SDS)-protein complexes were then separated and baseline resolved in 8 s using a 5% linear polyacrylamide/0.1% (w/v) SDS-based separation buffer, 3 mm separation channel, and 303 V cm electric field. 

L. Gaillon, S. Cozette, J. Lelievre and R. Gaboriaud, New pseudo-stationary phases for electrokinetic capillary chromatography. Complexes between bovine serum albumin and sodium dodecyl sulfate, J. Chromatogr. A, 876, 169-182, 2000. 

Protein-polyphenols interaction has been probed by various analytical methods that include high performance liquid chromatography (HPLC) [4,8], sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [9], binding assay [2,10-11], nephelometry [12-15], and nuclear magnetic resonance (NMR) spectrometry [1,3,16,17], but with protein models that were often far from giving a realistic representation of salivary proteins bovine serum albumin (BSA) [18,19], gelatin [20,21], casein [1,22], polyvinylpolypyrrolidone (PVPP) [23,24], bradykinin [25,26], or diverse synthetic proteins [27,28]. 

S. Shinagawa, K. Kameyama and T. Takagi, Effect of Salt Concentration of Buffer on the Binding of Sodium Dodecyl Sulfate and on the Viscosity Behavior of the Protein Polypeptide derived from Bovine Serum Albumin in the Presence of the Surfactant, Biochim. Biophys. Acta, 1161 79 (1993). 

Fig. 6. Comparative analyses of endogenous (ADPR)n nonhistone proteins from different hepatic tissues. The endogenous poly(ADPR) nonhistones were isolated from different types of untreated liver cells. Electrophoretic analyses were performed on 7% polyacrylamide gels using the Tris/ phosphate buffer system [11 at pH 6.0 in the presence of 0.1% sodium dodecyl sulfate and 6 M urea. Gels were stained with silver stain (Bio-Rad, Miinchen). Samples applied to the gel correspond to the yield obtained from 10 cells./ip Yoshida hepatoma AH 1914 nh normal hepatocytes after 44 h in primary culture It normal liver tissue cells from whole animals ma marker proteins from top to bottom Myosin (200 kD), ]3-galactosidase (116 kD), phosphorylase b (92 kD), bovine serum albumin (66 kD)...

Bovine serum albumin (Cat. No. ORHD 20/21) was from Behringerwerke AG, Dulbecco s modified Eagle s medium (Cat. No. 07402100) from Gibco-BRL Life Technologies, and sodium dodecyl sulfate (Cat. No. 20760) from Serva Feinbio-chemica GmbH Co KG. 

Fiq. 2. Precipitation, of horse serum albumin by sodium dodecyl sulfate as a function of detergent/protein weight ratio at pH 4.5 in 0.1 N sodium acetate buffer. Adapted from Putnam and Neurath (114). 

Fig. 3. Electrophoretic analysis of serum albumin—sodium dodecyl sulfate mixtures at 1°C. Figures between patterns represent experiment numbers. In all experiments 0.3% detergent was present except in experiment 1 which contained only pure protein. In experiments 2 to 6 the protein concentration decreased as follows 2.0%, 1.5%, 1.0%, 0.8% and 0.6%. Since all experiments were run for the same time (14,400 sec.) at essentially the same field strength (4.7 v./om.), constancy of mobilities of components labeled A, 1,2, and D may be observed by comparing directly displacements of respective boundaries from the common origin (starting boundary characterized by the pair of vertical lines). Arrows denote direction of migration right, descending left, ascending boundary. From Putnam and Neurath (117).

The hybrid or complex nonchemical interactions are the basis for the construction of new artificial systems composed of certain proteins, such as P-lacto-globulin or serum albumin, with sodium dodecyl sulfate (SDS) to be used in systems in which electrostatic stabilization of O/W emulsions is essential. 

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