S-nitrosylation is light-sensitive, and light can also react with ascorbate to induce artifactual signals; therefore, all procedures involving detection or stimulation of S-nitrosylation were carried out under minimal light conditions using brown Eppendorf tubes and foil-covered polypropylene falcon tubes. No glassware/metalware was used to avoid contaminating metal species that could interfere with the reaction (40, 41). CysNO was prepared as previously described (42). Cys only was prepared as for CysNO using Bio Perfomance Certified (BPC)–grade water (W3513, Sigma-Aldrich) in place of NaNO2. Buffers were prepared as in (15) with additional buffers used as specified.

For neuronal samples, 3 × 10 cm plates of E17 cortical neurons per sample were treated as appropriate. For HEK experiments, a transfected 6 cm plate of HEK293T cells was treated per condition. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and harvested in cold HEN lysis buffer [100 mM Hepes, 1 mM EDTA, and 0.1 mM neocuproine (pH 8.0) with 0.2% NP-40]. Samples were then homogenized five times with a 25-gauge needle and centrifuged for 10 min at 10,000g at 4°C, and the supernatant was collected (neuronal samples contained ~1.2 mg of protein per three plates, and HEK293T cells contained 650 μg of protein per sample). Except for experiments that were lysed and blocked immediately in Fig. 2, low–molecular weight S-nitrosothiols were removed by acetone precipitation at −20°C for >1 hour, followed by centrifugation of protein pellets for 20 min at 2000g at 4°C, two washes with 70% acetone, and resuspension in 200 μl of HENS [100 mM Hepes, 1 mM EDTA, and 0.1 mM neocuproine (pH 8.0) with 1% SDS]. Samples were blocked by adding an equal volume of 2× methyl thiocysteine (MMTS) blocking buffer (2% MMTS and 5% SDS in HEN buffer) and incubated for 30 min at 50°C with intermittent vortexing. Protein pellets were precipitated and, as before, then washed three times with 70% acetone and resuspended in 220 μl of HEN lysis buffer. Sodium ascorbate was added to a final concentration of 50 mM, and samples were incubated for 5 min at room temperature (RT) with gentle mixing. HPDP-biotin (catalog no. 21341, Thermo Fisher Scientific) was added to 1 mM, and samples were rotated for 45 min, followed by acetone precipitation as above. After three washes with 70% acetone, samples were resuspended in 220 μl of HENS, and 400 μl of neutralization buffer (25 mM Hepes, 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, pH 7.5) was added. Samples were precleared by incubation with protein A beads for 1 hour at RT and then centrifuged at 500g for 5 min at RT, and the supernatant was removed. Total inputs (10%) were taken, and samples were incubated with 30 μl of bead volume of streptavidin agarose beads overnight, rotating at 4°C. Samples were then centrifuged at 500g for 5 min at 4°C and washed four times in wash buffer (neutralization buffer, 600 mM NaCl). Beads were dried using a 29-gauge needle, and 40 μl of 2× Western loading buffer [2% SDS, 2% beta mercaptoethanol, 20% glycerol, 100 mM tris (pH 6.8), and 0.008% bromophenal blue] was added. Samples were then boiled for 5 min and then centrifuged at 10,000g for 2 min at RT, and the supernatant was run on homemade 10% polyacrylamide gels or precast NuPAGE 4-12% bis-tris gels (NP0335). For biotin switch on treated cellular lysates, the above protocol was followed, except that the treatment took place after harvesting in samples made up to 500 μl with HEN buffer. In experiments using N-ethylmaleimide-cysteine (NEM) blocking (140 mM in HEN buffer with SDS at 2.44%), blocking was carried out for 45 min at 50°C.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.