Immunoprecipitation. For the detection of the constitutive glutathionylation of TF, solubilized monocytes were centrifuged 3 times at 3,000 g for 2 minutes at 4°C, and the supernatant was incubated with the monoclonal anti-GSH antibody (Virogen; or control IgG) overnight at 4°C. In other experiments, intact monocytes were incubated with anti-GSH antibody for 60 minutes at room temperature. Then, protein A Sepharose (Sigma-Aldrich; 50 μl) was added and the mixture incubated for 60 minutes at 4°C. Immunoprecipitated complexes were collected by centrifugation at 3,000 g for 2 minutes at 4°C. After washing 3 times with PBS, the pellets were incubated in SDS sample buffer (without reducing reagents) for 60 minutes at 20°C. After centrifugation, the supernatant was subjected to SDS-PAGE (7.5%), followed by electroblotting onto the nitrocellulose membrane. The membranes were then exposed to a monoclonal anti-TF antibody (or control IgG), followed by a horseradish peroxidase–conjugated anti-mouse IgG. The experiments were repeated 3 times.

TF glutathionylation. For the labeling of GSH with biotin, equimolar amounts (10 mM) of NHS-biotin (Pierce Biotechnology) and GSH dissolved in PBS were incubated at room temperature. The reaction was terminated after 1 hour, the residual biotin being quenched with ethanolamine (50 mM). To enable the protein incorporation of the labeled glutathione, the cells (or sTF) were incubated for 30–60 minutes with biotin-GSH (5 mM). Subsequently, the cells were washed and solubilized with 1% Triton X-100 in PBS. sTF was dialyzed using centricon filters. Then streptavidin beads (Sigma) were added to the biotinylated proteins of the cells (or sTF) and the mixture was incubated overnight. After sedimentation of the beads, they were washed twice with PBS containing 0.2% Triton X-100. The proteins were eluted in SDS sample buffer, separated by SDS-PAGE, transferred to nitrocellulose membranes, and the monoclonal anti-TF antibody was employed to detect the glutathionylated protein.

谷胱甘肽抗體Anti-Glutathione mAb操作使用文獻數據參考：

(A) Basal glutathionylation of cell TF. Solubilized monocytes were immunoprecipitated with anti-GSH antibody or with isotype control antibody, as described in Methods. This was followed by western blotting with control antibody or anti-TF antibody.

(C) Glutathionylation attenuates TF activation. Cell lysis increases procoagulant activity, relative to intact cells, which is completely inhibited by anti-TF antibody. Increasing concentrations of DTT suppress TF activity. Glutathionylation in lysed cells was induced by preincubation with DTT (0.1 mM), followed by incubation with GSH (0.1 mM plus 0.1 mM diamide; 15 minutes). This increased the density of the band representing glutathionylated TF in immunoblots of lysed monocytes by 5.5-fold. Procoagulant activity of the lysates was determined by coagulation factor concentrate. *P < 0.05 versus control. n = 3–9.

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