Adsorbed proteins identification

Vroman, L., Adams, A. L., Identification of Adsorbed Protein Films by... 

The capacity of the nanoparticles to adsorb proteins and to activate the complement in vivo after intravenous administration will influence the fate of the carrier and its body distribution. To approach this aspect, in vitro tests have been developed to investigate the profile of the type of serum proteins that adsorbed onto the nanoparticle surface after incubation in serum and to evaluate the capacity of the nanoparticles to induce complement activation. The analysis of the protein adsorbed onto the nanoparticle surface can be performed by 2D-polyacrylamide gel electrophoresis. This technique allows the identification of the proteins that adsorbed onto the nanoparticle surface. To evaluate modifications of the composition of the adsorbed protein with time, a faster method based on capillary electrophoresis can also be used. Finally, the activation of the complement produced by nanoparticles can be evaluated either by a global technique or by a specific method measuring the specific activation... 

It should be noted that during the preparation of any protein or peptide sample, some of the analyte will be permanently adsorbed to the walls of the vial, to packing material, or to any transfer device used such as a pipet tip. The loss of sample is of critical concern when working below approximately 100 fmole/pl. Protein adsorption by some vials is so great that the sample will not be observed in the mass spectrum. In addition, contamination from airborne protein particles, such as human or sheep keratin, can cause misleading results when working at these low levels. Covering your pipet tips and vials is recommended for protein identification work. 

Fig. 1. Identification of the endogenous brain GITl/p95-APPl complex by affinity chromatography on Racl-GTP columns. 100 tig of GST-Racl for each sample were adsorbed on 25 of glutathione-agarose beads. The adsorbed protein was loaded with GTP7S (GTP) or GDP/3S (GDP), washed, and each sample was then incubated for 1 h with rotation at 4° with 2 mg of lysate from E13 avian brain. After washing, samples were eluted for 1 h at room temperature with 8 M urea, and one third of the eluted fractions were loaded on a 8% acrylamide gel. 6 /rg of brain lysate (Lys), and eluate from beads loaded with GST-Racl and buffer only (last lane to the right) were included. After separation by SDS-PAGE, the gel was silver stained.

Figure 5.14 MALDI-ToF MS of three different plasma standards bovine, canine, and human on polyurethane membrane, (a) Low-molecular weight region, (b) High-molecular weight region, (c) Identification of adsorbed proteins in mass spectra compared with the literature values of proteins.

Bale MD, Wohlfahrt LA, Mosher DF et al (1989) Identification of vitronectin as a major plasma-protein adsorbed on polymer surfaces of different copolymer composition. Blood 74(8) 2698-2706... 

The wettability of sites where presumably antibody had been deposited on an antigenic film allowed rapid identification on proteins adsorbed on surfaces such as unoxidized metal or on others that were unfit for interference color or Coomassie Blue observation. Since all data confirmed those obtained by other means they will not be listed. Some details are of interest. Wherever water drops condensed and were allowed to evaporate, a dot of matter presumably transported by the moving air/water boundary was deposited in the center of each drop during evaporation. With reexposure to air saturated with water, condensation would start on each dot and result in a pattern identical to the first one. Coomassie Blue staining, or exposure to metal oxide suspensions 110), would show a reticulum of protein concentrated between the water drop sites. 

Analysis of in vivo-formed pellicle by a combination of electrophoretic separation and MALDI-TOF showed the presence of intact histatin 1, cystatin SN, statherin, lysozyme, albumin and amylase [15, 39], In addition, intact cytokeratins 13 and 15 and calgranulin B were identified as components of the salivary pellicle layer for the first time using MALDI-TOF mass spectrometry [15]. Calgranulin B has been shown to be a component of saliva and gingival crevicular fluid [15]. The identification of cytokeratins in the salivary pellicle layer points to the oral epithelium as one of the sources of proteins adsorbed on the tooth surface. 

The biological aids to purification of phages are of three types (1) those based on the specific adsorbability of the virus to host cells, (2) those based on the specific precipitability of phage or bacteria by anti-sera, and (3) those involving digestion with enzymes. The first two methods are not ordinarily available to the protein chemist. With phages they may be used either for the removal of contaminating substances or for the identification of the virus activity with material to be measured for isotope content. In isotopic experiments only a small fraction of the added tracer may appear in the virus hence, it is desirable to identify the virus activity with the isotope by more specific criteria than those based on size alone. 

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