48 h after transfection with siRNA, HT1080s were lysed in RIPA buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, 150 mM NaCl, and 50 mM Tris, pH 8) plus 1× protease inhibitors without EDTA (Roche). Cleared lysates were combined with an equal volume of 2× sample buffer, heated to 95°C for 5 min, resolved by SDS-PAGE on a 4–12% Tris-glycine polyacrylamide gel (Thermo Fisher Scientific), and transferred to nitrocellulose (0.2-µm pores; Thermo Fisher Scientific). Secondary antibodies used for Western blotting were: goat anti–rabbit IgG Alexa 680 and goat anti–mouse IgG Alexa 680 (Thermo Fisher Scientific). Blots were visualized on an Odyssey Scanner (LI-COR Biosciences). Western blots were quantified by normalizing the signal intensity of nesprin 3, vimentin, or lamin A to the corresponding GAPDH signal and determining the change in expression of the siRNA-mediated protein knockdown relative to the control.
Goat anti rabbit igg alexa 680
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-rabbit IgG Alexa 680 is a secondary antibody conjugated with the Alexa Fluor 680 fluorescent dye. It is designed for the detection of rabbit primary antibodies in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg alexa 680, by Thermo Fisher Scientific (3 mentions)
Tris glycine polyacrylamide gel, by Thermo Fisher Scientific (3 mentions)
Odyssey scanner, by LI COR (3 mentions)
Nitrocellulose membranes, by Thomas Scientific (2 mentions)
Nitrocellulose, by Thermo Fisher Scientific (1 mentions)
Anti gfap, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti map2, by Merck Group (1 mentions)
Anti sqstm1 p62, by Abcam (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti phospho sqstm1 p62 s349, by Abcam (1 mentions)
Goat anti rat alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Irdye 800 goat anti mouse igg, by LI COR (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti gapdh, by Abcam (1 mentions)
4 protocols using goat anti rabbit igg alexa 680
1
Western Blot Analysis of siRNA Knockdown
2
Western Blot Analysis of E-cadherin and Vimentin
3
Immunofluorescence and Western Blot Antibody Protocol
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In immunofluorescence experiments, the following antibodies and dilutions were used: anti-Phospho SQSTM1/p62 (S349) (Abcam, Cambridge, UK, cat # ab211324) 1:100; anti-SQSTM1/p62 (Abcam, cat # ab56416) 1:50; anti-β-Tubulin III (Merck Millipore, Burlington, MA, USA cat # T2200) 1:500; anti-MAP2 (1:500, Merck Millipore, cat # M9942) 1:500; anti-GFAP (Thermo Fisher Scientific, Waltham, MA, USA, cat # 13-0300), 1:800, donkey anti-rabbit-IgG Alexa Fluor 488 (Thermo Fisher Scientific, cat # R37118) 1:1000; donkey anti-mouse-IgG Alexa Fluor 594 (Thermo Fisher Scientific, cat # A-21203, 1:1000); goat anti-mouse IgG1 CF 568 (Merck, cat # SAB4600313 1:1000); goat anti-rat Alexa Fluor 647 (Thermo Fisher Scientific, cat # A21247, 1:1000); and DAPI (4′,6-diamidino-2-phenylindole Merck Millipore, cat #D9542) 1:5000. In Western blotting experiments, the following antibodies and dilutions were used: anti-Phospho SQSTM1/p62 (S349) (Abcam cat # ab211324) 1:2000; anti-SQSTM1/p62 (Abcam cat # ab56416) 1:2000; Anti-GAPDH (Abcam cat # ab8245); 1:5000; anti-phospho-Akt (Ser473 D9E Cell Signaling, Danvers, MA, USA, cat #4060) 1:2000; anti-Akt (Cell Signaling, cat # 9272, 1:1000); goat anti-mouse IgG IRDye 800(Li-Cor, Lincoln, NE, USA, cat # 926-32210) 1:5000; and goat anti-rabbit-IgG Alexa 680 (Thermo Fisher Scientific cat # A21076) 1:5000.
4
Western Blot Analysis of Epithelial-Mesenchymal Transition
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After treatment, MDCKs were lysed using RIPA buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 50mM Tris, pH8) supplemented with 1X protease inhibitors without EDTA (Roche). Cleared lysates collected were combined with an equal volume of 2X reducing sample buffer, heated to 95°C for 5 min, resolved on 4-12% Tris-glycine polyacrylamide gel (Thermo Fisher Scientific), and then transferred to nitrocellulose membranes containing 0.45 µm pores (Thomas Scientific). Secondary antibodies: goat anti-rabbit IgG Alexa 680 and goat anti-mouse IgG Alexa 680 (Thermo Fisher Scientific). Blots were scanned using Odyssey Scanner (LI-COR Biosciences). Western blots were quantified by normalizing the signal intensity of the E-cadherin or vimentin bands to the corresponding GAPDH signal following background subtraction.
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