Anti-eNOS antibody [M221] (ab76198)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Mouse (1:4000) --To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Mouse (1:4000) --To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Cells were lysed in buffer containing NaCl (150 mM), Tris-HCl (50 mM), EDTA (1 mM), NaF (1 mM), Na3VO4 (1 mM), Nonidet P40 (1%), sodium deoxycholate (0.25%) and proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, USA). Proteins were separated by NuPAGE 4–12% Bis-Tris gel (Invitrogen) electrophoresis and electro transferred (Trans-blot SD, Bio-Rad, Hercules, USA) to nitrocellulose membranes (Bio-Rad). As molecular weight marker, Precision Plus Protein Standard (BioRad, Cat. No. 161-0374) was applied. After incubation for 1 h in blocking-solution containing 5% dry milk in Tris-buffered saline, membranes were washed in Tris-buffered saline and incubated overnight at 4 °C with the antibodies: monoclonal anti-total eNOS antibody (diluted 1:4000, Abcam, cat-No. ab76198). After washing, blots were incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. As a HRP substrate ChemiGlow (Alpha Innotec Corporation, San Leandro, USA) was used and recorded by a CCD-camera system (Fluor Chem FC3, CellBiosciences, Santa Clara, USA). Wherever possible, quantitative comparisons between samples have been done on the same blot, in cases where a comparison was not possible on one blot, different blots were used, with samples derived from the same experiment, analysed in the same concentration and in parallel.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-eNOS antibody [M221] (ab76198) below.

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