Cells were lysed using RIPA lysis buffer (Beyotime, China), and the protein concentrations were measured using the Bicinchoninic Acid Protein Assay Kit (Beyotime, China). The proteins were separated on a 10% SDS-polyacrylamide gel and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Germany), as described previously28 (link). Following 2 h of blocking with TBST supplemented with 5% non-fat dried milk, the membranes were incubated overnight with specific primary antibodies at 4 °C. Anti-PI3K (1:1000, Abcam), anti-Bcl-2 (1:1000, Abcam), anti-AKT (1:2000, Abcam), anti-pAKT (1:500, Abcam), anti-BAX (1:1000, Abcam), anti-LC3B-I (1:500, Abcam), anti-p62 (1:2000, Abcam), anti-mTOR (1:1000, Abcam), anti-cleaved caspase 3 (1:1000, Abcam), and anti-pro-caspase 3 (1:1000, Abcam) were used as primary antibodies. A next step involved washing the membranes, exposing them to peroxidase-conjugated secondary antibodies, and developing them with ELC (Amersham Pharmacia, UK); incubation at room temperature for another 60 min was performed with goat anti-rabbit IgG (1:5000) or goat anti-mouse IgG (1:5,000). Thermo provided the software and hardware for measuring immunoreactivity. The Super Signal West Femto Maximum Sensitivity Substrate Kit was used on a C-Digit Blot Scanner to measure immunoreactivity.
Goat anti rabbit igg
Manufactured by Cytiva
Sourced in United Kingdom, United States
Goat anti-rabbit IgG is a secondary antibody that binds to rabbit immunoglobulin G (IgG) antibodies. It is used in various immunological techniques, such as Western blotting, ELISA, and immunohistochemistry, to detect and visualize the presence of rabbit-derived primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti akt, by Abcam (1 mentions)
Anti p akt, by Abcam (1 mentions)
Anti pro caspase 3, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Goat anti mouse igg, by Cytiva (1 mentions)
Anti p62, by Abcam (1 mentions)
Anti mtor, by Abcam (1 mentions)
Anti pi3k, by Abcam (1 mentions)
H 7650, by Hitachi (1 mentions)
H 7100, by Hitachi (1 mentions)
Normal goat serum (ngs), by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Rabbit anti alix, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Polyscreen, by PerkinElmer (1 mentions)
4 protocols using goat anti rabbit igg
1
Western Blot Analysis of Signaling Proteins
2
Immunolabeling Mycoplasma in Vero Cells
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Vero cells infected with M. salivarium were subjected to immunoelectron microscopy by ultracryotomy-immunolabeling methods, as described by Akagi et al. (23 (link)). Briefly, M. salivarium-infected Vero cells were collected by centrifugation and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at room temperature for 2 h. The pellet was immersed in 0.1 M PB containing 30% sucrose at room temperature for 30 min and transferred to 20% polyvinylpyrrolidone and 1.84 M sucrose in 0.1 M PB at room temperature for 2 h. After infusion, the sample was frozen with liquid propane at −185°C. The frozen sample was then cut into ultrathin cryosections, and the sections were transferred to carbon-/formvar-coated grids (#100) (Nisshin EM, Tokyo, Japan). After blocking with 10% normal goat serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 0.1 M TBS for 1 h, the sections were incubated in 0.1 M Tris-buffered saline (TBS) containing the PAb (1:2000) at room temperature for 1 h, then at 4°C for 2 days, and finally at room temperature for 1 h. The sections were incubated for 2 h at room temperature in 0.1 M TBS containing the colloidal gold (φ: 10 nm)-conjugated secondary antibody (1:200) goat anti-rabbit IgG (Amersham Pharmacia Biotech). Immunolabeled sections were observed under H-7100 and H-7650 electron microscopes (Hitachi High-Technologies, Tokyo, Japan).
3
Chemokine receptor expression in cancer cell lines
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CAL-27, UM-1 and TCA-8113 cell lysates were electrophoresed on a 10% acrylamide gel and immunoblotted onto nitrocellulose membranes. The nitrocellulose membranes were washed, blocked with 5% nonfat dry milk in tris-buffered saline (TBS) for 90 min at 25°C and then incubated overnight with rabbit polyclonal antibodies for C-C chemokine receptor type 5 (CCR5; 1:1,000), C-C chemokine receptor type 6 (CCR6; 1:1,000) and C-X-C motif chemokine receptor 3 (CXCR3; 1:1,000), following the manufacturer’s instructions (Bioss Inc., Beijing, China). Membranes were washed and then incubated with goat anti-rabbit IgG for 1 h, before they were visualized by enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ, USA) on an X-ray film.
4
Protein Immunoblotting Analysis Protocol
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Analyzed proteins were quantified and used for immunoblotting analyses. The equal quantified proteins or equal volumes of immunoprecipitated proteins were analyzed by SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes (PolyScreen, PerkinElmer). Transferred antigens were incubated with the following antibodies: mouse anti-galectin-3, rabbit anti-Alix, mouse anti-HIV-1 p24 (Millipore, Burlington, MA, USA), mouse anti-β-actin (Sigma, St. Louis, MO, USA), or rabbit anti-α-tubulin (Epitomics, Burlingame, CA, USA), and held for 1 h at 37 °C. After three washes with 1x phosphate-buffered saline with 0.1% Tween® detergent (PBST), membranes were incubated with horseradish peroxidase (HRP)-conjugated antibodies (goat anti-mouse IgG, goat anti-human IgG, or goat anti-rabbit IgG) (Amersham Biosciences, Charlemagne, UK) for 1 h at 37 °C. Hybridized protein bands were created with an Immobilon™ Western Enhanced Chemiluminescence (ECL) protein detection system (Millipore, Burlington, MA, USA).
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