Transduced primary cortical neurons (24-well, 7 d after viral delivery) or transduced tissue samples (4 weeks after viral delivery) were lysed in 50 μl of ice-cold RIPA buffer (Cell Signaling) containing 0.1% SDS and proteases inhibitors (Roche, Sigma). Cell lysates were sonicated for 5 min in a Bioruptor sonicater (Diagenode) and protein concentration was determined using the BCA Protein Assay Kit (Pierce Biotechnology, Inc.). Protein lysates were dissolved in SDS-PAGE sample buffer, separated under reducing conditions on 4–15% Tris-HCl gels (Bio-Rad) and analyzed by western blotting using primary antibodies: rabbit anti-Dnmt3a (H-295, Santa Cruz, 1:500), mouse anti-Dnmt1 (60B1220.1, Novus Biologicals, 1:800), rabbit anti-MeCP2 (07-013, Millipore, 1:400), mouse anti-HA (6E2, Cell Signaling, 1:400) rabbit anti-Tubulin (AA2, Sigma, 1:10,000) followed by secondary anti-mouse and anti-rabbit HRP antibodies (Sigma-Aldrich, 1:10,000). GAPDH was directly visualized with rabbit HRP coupled anti-GAPDH antibody (14C10, Cell Signaling, 1:10,000). Tubulin or GAPDH served as loading control. Blots were imaged with ChemiDoc MP system with ImageLab 4.1 software (Bio-Rad), and quantified using ImageJ software 1.48 h.
Anti mouse and anti rabbit hrp antibodies
Manufactured by Merck Group
Anti-mouse and anti-rabbit HRP antibodies are laboratory reagents used in various immunodetection techniques. They serve as secondary antibodies that bind to primary antibodies raised against mouse or rabbit proteins, and are conjugated with the enzyme Horseradish Peroxidase (HRP). These antibodies can be used to detect and visualize the presence of target proteins in samples, such as in Western blotting, ELISA, and immunohistochemistry applications.
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Lab products found in correlation
Tris hcl gel, by Bio-Rad (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Mouse anti ha 6e2, by Cell Signaling Technology (1 mentions)
Rabbit α ha, by Cell Signaling Technology (1 mentions)
2 protocols using anti mouse and anti rabbit hrp antibodies
1
Quantitative Analysis of Epigenetic Regulators
2
Antibody Profiling for Cellular Localization
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The primary antibodies used in this study were as follows: monoclonal mouse α-Ty (hybridoma BB2, 1:10 IFA, 1:10 WB), mouse α-HA 16B12 (Thermo Fisher; 1:1,000 WB), rabbit α-HA (Cell Signaling Technology; 1:400 IFA, 1:500 WB), mouse α-cMyc (Thermo Scientific; 1:100 IFA, 1:500 WB), rat α-cMyc (Abcam; 1:200 IFA), mouse α-Rab11A (S. Marion lab; 1:250 IFA), α-actin (1:10 IFA, 1:10 WB), α-GAP45 (1:10,000 IFA), α-catalase (1:2,000 WB), α-SAG1 (a generous gift from J.-F. Dubremetz, 1:5 IFA), α-GRA1 (1:3,000 WB; Anawa) α-GRA3 (a generous gift from J.-F. Dubremetz, 1:100 IFA), and α-MIC2 (a generous gift from V. Carruthers; 1:10 IFA, 1:10 WB, α-ARO; 1:1,000 IFA). The secondary antibodies used in this study were anti-mouse and anti-rabbit HRP antibodies (Sigma), Alexa Fluor 680-conjugated goat anti-rabbit antibodies and anti-mouse IgG antibodies, Alexa Fluor 488-conjugated goat anti-rabbit and anti-mouse IgG antibodies, and Alexa Fluor 594-conjugated goat anti-rabbit and anti-mouse IgG antibodies (1:20,000 IFA, 1:20,000 WB; Thermo Fisher).
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