After tissue homogenization, protein samples (20 µg) were loaded onto 12% polyacrylamide gels. After transfer, the membranes were treated with 0.25% glutaraldehyde at room temperature for 10 min in 0.2% Tween 20/tris-buffered saline (TTBS) to crosslink tissue proteins that might destroy or mask immunogenic epitopes within the tissue [31 (link),32 (link)]. The membranes were then blocked with 5% skimmed milk and incubated with a rabbit monoclonal antibody against STMN1 (ab52630; Abcam) diluted 1:50,000, and with a rabbit polyclonal antibody phosphorylated at S-16 (3353; Cell Signaling Technology, Danvers, MA, USA) and diluted 1:1000 at 4°C overnight. After washing five times with TTBS, the membranes were incubated with peroxidase-labeled goat anti-rabbit IgG (H + L) (Vector Laboratories, Burlingame, CA, USA) diluted 1:5000 at room temperature for 2 h.
Peroxidase labeled goat anti rabbit igg h l
Manufactured by Vector Laboratories
Sourced in United States
Peroxidase-labeled goat anti-rabbit IgG (H + L) is a secondary antibody used for detection in immunoassays. It is produced by immunizing goats with rabbit IgG and then labeling the resulting antibodies with the enzyme peroxidase.
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Lab products found in correlation
3 protocols using peroxidase labeled goat anti rabbit igg h l
1
Western Blot Analysis of STMN1 and Phosphorylated STMN1
2
Quantification of D2 receptor isoforms
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Two isoforms of the D2R, D2L and D2S have been analyzed previously and a few modifications were observed [33 (link),34 (link)]. A detailed description of this analysis was reported in our previous study [26 (link)]. Glutaraldehyde-treated membranes were incubated in 5% skimmed milk overnight at 4 °C with rabbit polyclonal antibodies against D2L (1:2000) and D2S (1:5000) (AbClon, Inc., Seoul, Korea). After the membranes were washed three times, the primary antibodies were detected using peroxidase-labeled goat anti-rabbit IgG (H + L) (1:3000 for D2L and 1:5000 for D2S; Vector Laboratories) for 2 h at room temperature (25 °C).
3
SDS-PAGE and Western Blot Analysis
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blots were performed according to standard procedures. The cells were lysed in lysis buffer [50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.6 % octylphenoxypolyethoxyethanol (IGEPAL CA-630), 1 mM EDTA] and separated on a 7.5 or 10 % Tris–glycine gel, and then transferred to a nitrocellulose membrane. Blots were blocked with 5 % skim milk in PBS and incubated with a primary antibody and washed three times with PBS-T. Next, the blots were incubated with peroxidase-labeled horse anti-mouse IgG (H + L) or peroxidase-labeled goat anti-rabbit IgG (H + L) (Vector Laboratories) and washed with three changes of PBS-T. They were then visualized by using ImmunoCruz™ Western Blotting Luminol Reagent (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Images were captured with a cooled CCD camera.
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