Non-reduced condition

Fig. 1.4 Protein blot analysis of C5-1 assembly in agroinfiltrated alfalfa leaves. Total leaf soluble proteins, extracted 4 days after infiltration were separated by SDS-PAGE under non-reducing conditions and blotted onto a PVDF membrane. Polyclonal antimouse IgGs were used for detection. Purified C5-1 was mixed with total soluble proteins from control infiltrated alfalfa leaves and loaded as a standard.

Fig. 8.1 Western blot analysis of transgenic lines showing the expression of an assembled monoclonal antibody in transgenic chloroplasts. Lane 1 Extract from a chloroplast transgenic line, Lane 2 Extract from an untransformed plant. Lane 3 Positive control (human IgA). The gel was run under non-reducing conditions. The antibody was detected with an AP-conjugated goat anti-human kappa antibody.

FIGURE 6 CE-SDS separations under non-reduced conditions of a 5-TAMRA SE-labeled rMAb sample showing tailing of the main peak. Electrophoretic conditions as in Figure 5. 

Examine by SDS-PAGE (2.2.31). The test is performed under both reducing and non-reducing conditions, using resolving gels of 14% acrylamide and silver staining as the detection method. 

Sample buffer (non-reducing conditions). Mix equal volumes of water R and concentrated SDS-PAGE sample buffer R. 

The electropherogram obtained with test solution (a) under non-reducing conditions may show, in addition to the principal band, less intense bands with molecular masses higher than the principal band. No such band is more intense than the principal band in the electropherogram obtained with reference solution (d). (1%) and not more than 3 such bands are more intense than the principal band in the electropherogram obtained with reference solution (e) (0.2%). 

Disulphide bonding. The two principal caseins, asl and / , contain no cysteine or cystine but the two minor caseins, as2 and k, each contains two cysteines per mole which normally exist as intermolecular disulphide bonds. Under non-reducing conditions, ocs2-casein exists as a disulphide-linked dimer (previously known as as5 casein) while K-casein exists as a series of disulphide-linked molecules ranging from dimers to decamers. 

EPR spectra of CO oxidoreductase under non reducing conditions showed a spectrum at g = 2.21, 2.11, and 2.02, which, by analogy with spectra observed in nickel-containing hydrogenases, was attributed to Ni(III) (96). The spectrum was of low intensity and it was not established whether it represents an active state of the enzyme. 

Once the structural features of a reference standard of the desired protein have been well characterized, lot-to-lot confirmation of identity can be conducted using a carefully selected group of tests, wherein the lot undergoing analysis is compared to the reference standard. Tests commonly employed for this purpose are listed in Table III. Peptide mapping is perhaps the most powerful and universally used technique since it provides relatively specific confirmation of correct primary sequence and, when non-reducing conditions are employed, can be used to confirm correct disulfide bond formation. Tertiary structure is difficult to address directly on a routine (lot-to-lot) basis, and the presence of correct biological activity is often used as evidence that the correct tertiary structure is maintained. 

This test separates proteins according to their molecular weight (Laemmli, 1970). Firstly, the sample is denatured in a detergent, which breaks the non-covalent intra- and inter-molecular links of the proteins, and then it is separated electrophoretically by a polyacrylamide gel. The electrophoretic separation must be performed under reduced and non-reduced conditions to determine the existence of impurities with the same molecular weight. 

The method under non-reduced conditions is normally used to estimate the aggregation and/or oligomerization of the protein but only aggregates or oligomers that are stable in the presence of SDS will be detected and under the conditions used for the sample preparation and electrophoresis. 

Figure 5-4. Identification of disulphide bonds in proteins by 2-dimensional SDS-PAGE. Proteins are cleaved into fragments either enzymatically or by chemical treatment, and the resulting fragments are separated by SDS-PACE under non-reducing conditions. The gel is then incubated with performic acid which causes...

All sample proteins were run on either 12% or 14% pre-cast Novex 1.0 mm, 10 well gels under non-reducing conditions according to Laemmli (7). Samples were immediately electroblotted to Immobilon-P PVDF membrane using a semi-dry (MilliBlot-SDE) electroblotter essentially quantitiatively (8). After blotting, PVDF was washed briefly in HPLC water and stained with 0.05% Brilliant Blue-G Coomassie (BB-G) /20% methanol /0.5% acetic acid or Amido Black (2). The membrane was kept wet and not allowed to dry (9). Enzymatic digestion was performed as described (2) with all digestion and extraction buffer volumes reduced to 25 pL. 

The whole virus modification with the thiuram disulfides. HIV-1 (MN) (equivalent of 1 mg p24 CA) in 20 ml was treated at 37 C with 50 mM test compoimds for 1 h in sodium phosphate buffer, pH 7. Samples were centrifuged for 1 h at 17,000 g at 4 °C to pellet the virus and remove the drug. Samples for electrophoresis were run under non-reducing conditions. The gels (4-20%) were supplied by NOVEX (San Diego, California). Then proteins were transferred onto PVDF membranes (Towbin et. al., 1979), stained with 0.5% (w/v) Ponceau S and detected by... 

In order to analyze the action of the thiuram disulfides on NC protein in whole virus, lOOOx concentrated cell-free HIV-l(MN) was incubated with 50 mM of thiuram disulfides for 60 min. The virus was then pelleted by centrifugation to remove reagents and was analyzed by western blot analysis with antibody against p7 (Fig. 6). Under non-reducing conditions the NC protein of untreated virus is a mixture of monomers, dimers, trimers and tetrameters (see Fig. 6, HIV-1 lane). These virus treated with thiuram disulfides showed NC antigen migrating above 200 kDa marker and in some cases the monomeric form of NCp7 was completely absent (tetraethyl-thiuram, tetraisopropylthiuram and dicyclopentamethylene-... 

Laemmli (12) gels (14%) were run in non-reducing conditions and stained with Coomassie Blue for detection of proteins as described (13). 

The copper can be reversibly removed from the active site by reaction with diethyldithiocarbamate under non-reducing conditions [86-88], or by cyanide after reduction by dithionate to Cu(I) [78]. The catalytic activity of the enzyme can be restored with high yield by addition of free Cu(II) ions to the apoenzyme [78,86]. In the case of pea seedling amine oxidase, addition of other bivalent metal ions does not lead to reactivation [86]. However the activity can be partially restored (from 15%) for the amine oxidase from bovine plasma by adding Co(II) [89]. Addition of Co(II) and Ni(II) can restore the original spectrum of the native enzyme with bovine serum amine oxidase reduced by dithionate [90]. 

The absorption spectrum of the copper amine oxidases shows a characteristic broad band at around 500 nm (460-510 nm) that confers a typical pink or yellow-pink color to highly purified enzyme preparations [10,12,32]. Absorption in the visible region is caused by the presence of the quinone cofactor and thus is not affected by removal of copper from the enzyme under non-reducing conditions [12,32], The quinone cofactor shows emission of fluorescence when excited at 280 and 365 nm [41,106]. Electron transition of Cu(II) is seen in circular dichroism spectra at 600-800 nm [12,32,107]. Under aerobic conditions, addition of the substrate leads to a temporary bleaching of the pink color of the enzyme, which is restored after complete consumption of the substrate. However, anaerobic conditions lead to the formation of a stable yellow intermediate... 

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