Immunoblot analysis was performed as previously described [4 (link)]. Briefly, cell lysates were prepared in lysis buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1% NP-40, 1% sodium deoxycholate) with 1 mM PMSF, 1 μg/μL aprotinin, and 1 μg/μL leupeptin as protease inhibitors and incubated on ice for 30 min. Total cell lysates (10 μg) were subjected to SDS-PAGE, followed by immunoblot detection with anti-Eea1 (1:1000, #MA5-14794, Invitrogen), anti-Hmox1 (1:1000, #5061, Cell Signaling Technology, Danvers, MA, USA), anti-Nqo1 (1:1000, #ab34173, Abcam, Cambridge, UK), anti-LC3B (1:1000, #2775, Cell Signaling Technology), anti-Ub(P4D1) (1:500, #8017, Santa Cruz Biotechnology, Dallas, TX, USA), anti-α-tubulin (1:500, #32293, Santa Cruz Biotechnology), or anti-β-actin antibodies (1:500, Santa Cruz Biotechnology). Based on the type of primary antibody, the appropriate HRP-conjugated goat anti-mouse or anti-rabbit IgG (1:10,000; Enzo Life Sciences) was used.
Hrp conjugated goat anti mouse or anti rabbit igg
Manufactured by Enzo Life Sciences
HRP-conjugated goat anti-mouse or anti-rabbit IgG is a secondary antibody conjugated with horseradish peroxidase (HRP). It can be used to detect and quantify mouse or rabbit primary antibodies in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin antibody, by Santa Cruz Biotechnology (2 mentions)
Anti tuj1, by Merck Group (2 mentions)
Anti notch3, by Santa Cruz Biotechnology (2 mentions)
Hrp conjugated donkey anti goat igg, by Santa Cruz Biotechnology (2 mentions)
Anti α tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti ub p4d1, by Santa Cruz Biotechnology (1 mentions)
Anti nqo1, by Abcam (1 mentions)
Anti eea1, by Thermo Fisher Scientific (1 mentions)
Anti hmox1, by Cell Signaling Technology (1 mentions)
Anti lc3b, by Cell Signaling Technology (1 mentions)
Anti ub, by Merck Group (1 mentions)
Anti cc3, by Merck Group (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Anti neun, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti casp3, by Cell Signaling Technology (1 mentions)
Fusion solo s image analyzer, by Vilber (1 mentions)
Phosphatase inhibitor mixture, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
4 protocols using hrp conjugated goat anti mouse or anti rabbit igg
1
Immunoblot Analysis of Cellular Proteins
2
Immunoblot Analysis of Cellular Proteins
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Immunoblot analysis was carried out as previously described with slight modifications12 (link). Briefly, cell lysates were prepared in immunoprecipitation (IP) buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1% NP-40, 1% sodium deoxycholate with 1 mM PMSF, 1 μg/μl aprotinin, and 1 μg/μl leupeptin as protease inhibitors) and incubated on ice for 30 min. Total cell lysates (15 μg) were subjected to SDS-PAGE, followed by immunoblot detection with anti-Tuj1 (1:1,000, Millipore), anti-Ub (1:1,000, Millipore), anti-Notch3 (1:200, Santa Cruz Biotechnology), anti-CC3 (1:1,000, Millipore) or anti-β-Actin antibody (1:2,000, Santa Cruz Biotechnology). Based on the types of primary antibodies, the appropriate HRP-conjugated goat anti-mouse or anti-rabbit IgG (1:10,000, Enzo Life Sciences) or HRP-conjugated donkey anti-goat IgG (1:5,000, Santa Cruz Biotechnology) was used.
3
Immunoblot analysis of neural markers
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For immunoblot analysis, cell lysates were prepared in hypotonic buffer and processed as previously described28 (link). Briefly, total cell lysates (15 μg) were subjected to SDS-PAGE, followed by immunoblot detection with anti-TUJ1 (1:1,000, Millipore), anti-GFAP (1:1,000, Millipore), anti-NeuN (1:500, Millipore), anti-CASP3 (1:3,000, Cell Signaling), anti-Notch3 (1:200, Santa Cruz Biotechnology), or anti-β-actin antibody (1:10,000, Sigma-Aldrich). Based on the types of primary antibodies, the appropriate HRP-conjugated goat anti-mouse or anti-rabbit IgG (1:10,000, Enzo Life Sciences) or HRP-conjugated donkey anti-goat IgG (1:5,000, Santa Cruz Biotechnology) was used.
4
Western Blotting Protein Detection
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For western blotting, cells were directly lysed in Laemmli buffer (50 mM Tris–HCl (pH 6.8), 4% SDS, 10% glycerol, and 5% β-mercaptoethanol) supplemented with a protease inhibitor cocktail (Sigma) and phosphatase inhibitor mixture (Roche). The total protein lysates were incubated on ice for 10 min and then heated at 99 °C for 10 min. The cell supernatants were obtained by centrifugation at 13,500 rpm for 15 min at 4 °C. Proteins were separated electrophoretically on a 10% or 15% SDS–PAGE and transferred onto a 0.45μm pore size nitrocellulose membrane (Cytiva). The membrane was incubated for 18 h with antibodies in 3% BSA (Hanlab) in TBS‐T (150 mM NaCl, 20 mM Tris–HCl (pH 8.0), and 0.05% Tween‐20) at 4 °C, followed by incubation with HRP‐conjugated goat anti-mouse or anti-rabbit IgG (Enzo Life Sciences) in 5% skim milk (Biopure) in TBS‐T at room temperature for 2 h. Proteins were visualized with ECL western blotting reagent using a Fusion Solo‐S image analyzer (Vilber). All images were quantified using ImageJ software.
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