Osteoclast lysates were prepared in lysis buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% IGEPAL CA-630, 2 mM EDTA, 50 mM NaF, 10% glycerol, protease inhibitor cocktail). Culture supernatants were collected to assess the secretion of Cathepsin K. Protein concentrations were determined with Bradford. After resolution by SDS-PAGE, proteins were transferred to PVDF-FL membranes (Immobilon-FL Transfer Membrane) and analyzed by immunoblot. Primary antibodies were: TUBB6 (1:1000), ARHGAP10 (ProteinTech S5136AP, 1:1000), GAPDH (CellSignaling #2118, 1:5000), Sigma antibodies EB1 (E3406, 1:2500), K40-acetylated tubulin (T6793, 1:1000) and actin (A2103, 1:1000), Abcam antibodies Vinculin (ab108620, 1:5000), CLASP1 (ab108620, 1:5000) and Histone H3 (ab1791, 1:1000), total β tubulin (Developmental Studies Hybridoma Bank E7, 1:1000), Santa Cruz Biotechnologies antibodies TUBB5 (SAO.4G5, sc-58884, 1:5000) and Cathepsin K (E-7, sc-48353, 1:500). Secondary antibodies were: Goat-anti-mouse Dylight680 (Invitrogen, 35518), Goat-anti-mouse Dylight800 (Invitrogen, SA535521), Goat-anti-rabbit Dylight680 (Invitrogen, 35568), Goat-anti-rabbit Dylight800 (Invitrogen, SA535571). Signals were acquired using the Odyssey Infrared Imaging System (LI-COR Biosciences, United States).
Goat anti mouse dylight 680
Manufactured by Thermo Fisher Scientific
Goat anti-mouse DyLight 680 is a secondary antibody conjugated with the DyLight 680 fluorescent dye. It is designed for the detection and quantification of mouse primary antibodies in various immunological applications, such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit dylight 800, by Thermo Fisher Scientific (6 mentions)
Gapdh antibody, by Merck Group (3 mentions)
Odyssey imaging system, by LI COR (3 mentions)
Nupage, by Thermo Fisher Scientific (3 mentions)
Tubb6, by Proteintech (2 mentions)
Arhgap10, by Proteintech (2 mentions)
Goat anti mouse dylight 800, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Goat anti rabbit dylight680, by Thermo Fisher Scientific (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Lysis buffer, by Merck Group (2 mentions)
Socs3, by Cell Signaling Technology (2 mentions)
P stat3, by Santa Cruz Biotechnology (2 mentions)
G6pdh, by Merck Group (1 mentions)
H3k36me2, by Active Motif (1 mentions)
Na934, by Santa Cruz Biotechnology (1 mentions)
Sa5 10044, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit dylight 800, by Thermo Fisher Scientific (1 mentions)
H3k36me3, by Abcam (1 mentions)
H2ax ser 139, by Merck Group (1 mentions)
9 protocols using goat anti mouse dylight 680
1
Osteoclast Protein Expression Analysis
2
Osteoclast Protein Immunoblot Analysis
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Osteoclast lysates were prepared in lysis buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% IGEPAL CA-630, 2 mM EDTA, 50 mM NaF, 10% glycerol, protease inhibitor cocktail).
Protein concentrations were determined with Bradford. After resolution by SDS-PAGE, proteins were transferred to PVDF-FL membranes (Immobilon-FL Transfer Membrane) and analyzed by immunoblot. Primary antibodies were: TUBB6 (1:1000), ARHGAP10 (ProteinTech S5136AP, 1:1000), GAPDH (CellSignaling #2118, 1:5000), Sigma antibodies EB1 (E3406, 1:2500), K40-acetylated tubulin (T6793, 1:1000) and actin (A2103, 1:1000), Abcam antibodies Vinculin (ab108620, 1:5000), CLASP1 (ab108620, 1:5000) and Histone H3 (ab1791, 1:1000), total β tubulin (Developmental Studies Hybridoma Bank E7, 1:1000), preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. TUBB5 (SAO.4G5, Santa Cruz Biotechnologies sc-58884, 1:5000). Secondary antibodies were: Goat-anti-mouse Dylight680 (Invitrogen, 35518), Goat-anti-mouse Dylight800 (Invitrogen, SA535521), Goat-anti-rabbit Dylight680 (Invitrogen, 35568), Goat-anti-rabbit Dylight800 (Invitrogen, SA535571). Signals were acquired using the Odyssey Infrared Imaging System (LI-COR Biosciences, USA).
Protein concentrations were determined with Bradford. After resolution by SDS-PAGE, proteins were transferred to PVDF-FL membranes (Immobilon-FL Transfer Membrane) and analyzed by immunoblot. Primary antibodies were: TUBB6 (1:1000), ARHGAP10 (ProteinTech S5136AP, 1:1000), GAPDH (CellSignaling #2118, 1:5000), Sigma antibodies EB1 (E3406, 1:2500), K40-acetylated tubulin (T6793, 1:1000) and actin (A2103, 1:1000), Abcam antibodies Vinculin (ab108620, 1:5000), CLASP1 (ab108620, 1:5000) and Histone H3 (ab1791, 1:1000), total β tubulin (Developmental Studies Hybridoma Bank E7, 1:1000), preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. TUBB5 (SAO.4G5, Santa Cruz Biotechnologies sc-58884, 1:5000). Secondary antibodies were: Goat-anti-mouse Dylight680 (Invitrogen, 35518), Goat-anti-mouse Dylight800 (Invitrogen, SA535521), Goat-anti-rabbit Dylight680 (Invitrogen, 35568), Goat-anti-rabbit Dylight800 (Invitrogen, SA535571). Signals were acquired using the Odyssey Infrared Imaging System (LI-COR Biosciences, USA).
3
Western Blot Analysis of Noradrenergic Nuclei
4
Western Blot Analysis of Noradrenergic Nuclei
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Noradrenergic nuclei from the brainstem were homogenized in lysis buffer (Sigma Aldrich, St. Louis, MO) and protein concentrations were determined using BCA assay as previously described12 (link), 13 (link). 20μg of protein was loaded for each sample into SDS-PAGE gels (NuPAGE, Invitrogen, Carlsbad, CA). Proteins were then transferred onto PVDF membranes which were probed with antibodies including pSTAT-3 (1:1000; goat-polyclonal; Santa Cruz Biotechnology, Dallas, TX), SOCS-3 (1:1000; goat-polyclonal; Cell Signaling Technology, Danvers, MA), and GAPDH antibody (1:2000; mouse-monoclonal; Sigma-Aldrich, St. Louis, MO). After washing 3 times of 10 min each, the membranes were incubated in blocking solution containing goat anti-rabbit DyLight 800 and goat anti-mouse DyLight 680 secondary antibodies (1:5000; Thermo Fisher Scientific; Waltham, MA). Bands were visualized and analyzed using an Odyssey imaging system (Li-COR biosciences, Lincoln, NE).
5
Antibody Validation for Histone Modifications
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The following antibodies were used: Set2 (raised in lab, 1:5000), G6PDH (Sigma-Aldrich A9521, 1:100,000), H3K36me3 (Abcam 9050, 1:1000 for ECL, 1:2000 for LI-COR, and 2 μL for ChIP), H3K36me2 (Active Motif 38255, 1:1000 for ECL and 1:2000 for LI-COR), H3K36me1 (Abcam 9048, 1:1000 for ECL and 1:2000 for LI-COR), H3K79me3 (Abcam 2621, 1:2000), H3 (EpiCypher 13-0001, 1:1000) (Supplemental Fig. S1C ) (Abcam 12079, 1:1000; CST 14269, 1:2000 for LI-COR, and EMD MilliporeSigma 05-928 2 µL for ChIP) (Supplemental Fig. S1E ). Rabbit (Amersham NA934, Donkey anti-Rabbit), goat (Santa Cruz 2768, Rabbit anti-Goat), rabbit (Thermo Fisher Scientific SA5-10044, Donkey anti-Rabbit DyLight 800), and mouse (Thermo Fisher Scientific 35518, Goat anti-mouse DyLight 680) secondary antibodies were used at 1:10,000.
6
Comprehensive Antibody Profiling for Cellular Analysis
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The antibodies used in this study were as follows: TGN46 (Abcam; ab16052); CCM3 (Acris; AP26023PU-N); BrdU (BD Biosciences; 555627), Lamp-2 (BD Biosciences; 555803), p21CIP1 (F-5) (Santa Cruz; sc-6246), p16INK4 (BD Biosciences; 511325), p62 (BD Biosciences; 610832); GAPDH (Calbiochem; CB-1001); LC3B (Cell Signaling; 3868), mTOR (Cell Signaling; 2983); H2AX (Ser139) (Millipore; 05-636); IL-8 (500-P28) (PeproTech; 500-P28); NFκB p65 (Santa Cruz; sc-8008); p53 (DO-1) (Santa Cruz; sc-126); C/EBPβ (Santa Cruz; sc-150); and tubulin (T5168) (Sigma-Aldrich). The secondary antibodies used were as follows: goat anti-rabbit DyLight™ 800, goat anti-mouse DyLight™ 680 (Thermo Scientific); goat anti-mouse Alexa 488, goat anti-rabbit Alexa 488, goat anti-mouse Alexa 594, and goat anti-rabbit Alexa 546 (Molecular Probes).
All plasmids were constructed using standard molecular biology techniques.
All plasmids were constructed using standard molecular biology techniques.
7
Western Blot Analysis of pSTAT-3
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Noradrenergic nuclei from the brainstem were homogenized in lysis solution (Sigma Aldrich, St. Louis, MO) and protein concentrations were determined using a micro BCA assay (Pierce, Rockford, IL). Equal quantities of protein (20 µg) were loaded on to SDS-PAGE gels (NuPAGE, Invitrogen, Carlsbad, CA) and separated. Gels were blotted onto nitrocellulose membranes and the membranes were probed with pSTAT-3 antibody (1:1000; goat-polyclonal; Santa Cruz Biotechnology, Dallas, TX) and GAPDH antibody (1:2000; mouse-monoclonal; Sigma-Aldrich, St. Louis, MO). After washing, the membranes were incubated in blocking solution containing goat anti-rabbit DyLight 800 and goat anti-mouse DyLight 680 secondary antibodies (1:5000; Thermo Fisher Scientific; Waltham, MA). Bands were visualized using an Odyssey imaging system (Li-COR biosciences, Lincoln, NE).
8
Antibody Characterization and Immunoblotting Protocol
9
Western Blot Protein Detection Protocol
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Samples were lysed in RIPA buffer (Cell Signaling Technology), and proteins were separated under denaturing conditions on 4%–15% polyacrylamide gels (Bio-Rad). After samples were transferred to polyvinylidene difluoride membranes (PVDF, Bio-Rad), blots were blocked and then incubated with primary antibodies overnight at 4°C. Goat anti-mouse HRP (115-035-003; Jackson ImmunoResearch) and goat anti-rabbit HRP (111-035-003; Jackson ImmunoResearch) were used for blot visualization on a chemiluminescence imaging system (Bio-Rad). Goat anti-mouse Dylight 680 (35518; Thermo Fisher Scientific) was used for detection of primary antibody on an Odyssey CLx imaging system (LI-COR).
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