The following primary and secondary antibodies were used for immunoblotting: mouse monoclonal anti-MCL-1 (clone 10, sc-12756) purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA; rabbit monoclonal anti-BCL-2 (clone 50E3, #2870), rabbit monoclonal anti-BCL-XL (clone 54H6, #2764) purchased from Cell Signaling Technology, Inc., Danvers MA, USA; and mouse monoclonal anti-GAPDH (GTX3066) purchased from GeneTex, Inc., Irvine, CA, USA; goat anti-mouse (170-6516) or anti-rabbit IgG (H + L)-HRP conjugate (170-6515) purchased from BioRad, Hercules, CA, USA. For indirect immunofluorescence, mouse monoclonal anti-MCL-1 (clone 10, sc-12756) purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA and Alexa Fluor 568 goat anti-mouse (A-11031) secondary antibody obtained from Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA, were used.
Goat anti mouse 170 6516
Manufactured by Bio-Rad
Sourced in United States
Goat anti-mouse (170-6516) is a secondary antibody that recognizes mouse primary antibodies. It is used in various immunoassay techniques to detect and quantify mouse-derived target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit 170 6515, by Bio-Rad (3 mentions)
Mouse monoclonal anti gapdh gtx3066, by GeneTex (2 mentions)
Anti rabbit igg h l hrp conjugate 170 6515, by Bio-Rad (2 mentions)
Donkey anti goat sc 2020, by Santa Cruz Biotechnology (2 mentions)
Anti gfap, by Synaptic Systems (1 mentions)
Anti β actin, by Novus Biologicals (1 mentions)
Anti gephyrin, by Synaptic Systems (1 mentions)
Anti myc, by OriGene (1 mentions)
Anti rabbit af488, by Thermo Fisher Scientific (1 mentions)
Anti map2, by Merck Group (1 mentions)
Anti rabbit af568, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti psd95, by Abcam (1 mentions)
Anti gm130, by BD (1 mentions)
Anti p53 1c12 2524, by Cell Signaling Technology (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Nupage 4 12 gel, by Thermo Fisher Scientific (1 mentions)
8 protocols using goat anti mouse 170 6516
1
Immunoblotting and Immunofluorescence Protocols for MCL-1, BCL-2, and BCL-XL
2
Protein Immunoblotting Assay
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Twenty-five to fifty micrograms of protein lysates were analyzed by SDS polyacrylamide gel electrophoresis (PAGE). After transfer to a 0.45-µm nitrocellulose membrane (GE Healthcare, Amersham, Thermo Fisher Scientific) and blocking in 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.05% (v/v) Tween 20 (TBST) containing 2.5% (w/v) skim milk, specific proteins were identified using primary antibodies for 16 to 18 h at 4 ℃. A secondary antibody [donkey anti-goat (sc-2020; Santa Cruz), goat anti-rabbit (1706515; Bio-Rad), or goat anti-mouse (1706516; Bio-Rad)] conjugated to horseradish peroxidase, and a luminol enhancer detection system (32106, Pierce, Thermo Fisher Scientific) were used to visualize the proteins.
3
Characterization of zDHHC15 Protein Expression
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Primary antibodies used were: anti-zDHHC15 (Abcam, Cambridge, MA, #ab121203, 1:250, western blot), anti-zDHHC15 (Thermo Scientific, Waltham, MA, #PA39327, immunofluorescence, 1:100), anti-Flag (Sigma Aldrich, St Louis, MO, #F3165, 1:1000), anti-Myc (Origene, Rockville, MD, #TA150121, 1:1000), anti-β-actin (Novus Biologicals, Centennial, CO, #NB600-503, 1:1000), anti-PSD-95 (Abcam, Cambridge, MA, #ab2723, 1:500), anti-gephyrin (Synaptic Systems, Göttingen, Germany #147011, 1:500), anti-GAD65 (Synaptic Systems, Göttingen, Germany #198104, 1:500), anti-GFAP (Synaptic Systems, Göttingen, Germany #173004, 1:400), anti-MAP2 (Millipore, Burlington, MA, MB3418, 1:2000), anti-giantin (Synaptic Systems, Göttingen, Germany, #263004, 1:500), anti-GM130 (BD Biosciences, Franklin Lakes, NJ, #610822, 1:200). Secondary antibodies from Life Technologies (Carlsbad, CA,) used: (1:500), anti-mouse AF568 IgG2a (#A21134), anti-mouse AF647 IgG1 (#21240), anti-rabbit AF488 (#A11008), anti-rabbit AF568 (#A11011), anti-guinea pig AF633 (#A21105). HRP conjugated secondary antibodies were obtained from Bio-Rad (Hercules, CA): goat anti-mouse #170-6516 and goat anti-rabbit (#170-6515, 1:300). DAPI was used for nuclear staining (Life Technologies, Carlsbad, CA, #D1306, 1:1000).
4
Immunoblotting Antibody Detection Protocol
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For immunoblotting, the following primary and secondary antibodies were used: rabbit polyclonal p70 S6 kinase (#9202), rabbit monoclonal phospho-p70 S6 kinase (Thr389) (#9234), rabbit monoclonal AKT kinase (#4691), rabbit monoclonal phospho-AKT kinase (Ser473) (#9234) obtained from Cell Signaling Technology, Inc., Danvers MA, USA; p21waf1 (sc-56335) obtained from Santa Cruz Biotechnology, Inc., Dallas, TX, USA and mouse monoclonal anti-GAPDH (GTX3066) purchased from GeneTex, Inc., Irvine, CA, USA; goat anti-mouse (170-6516) or anti-rabbit IgG (H + L)-HRP conjugate (170-6515) purchased from BioRad, Hercules, CA, USA.
5
Immunoblotting Antibodies and Reagents
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Anti-ABCG2 (D5V2K XP®, #42078) and anti-p53 (1C12, #2524) monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). Anti-β-actin (C4, sc-47778) and anti-β-tubulin (D-10, sc-5274) monoclonal antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Anti-xanthine oxidase monoclonal antibody (EPR4605, ab109235) was purchased from Abcam (Cambridge, MA, U.S.A.) and anti-GLUT9 polyclonal antibody (PA5-22966) was purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Horseradish peroxidase-conjugated goat anti-rabbit (#1706515) and goat anti-mouse (#1706516) were purchased from Bio-Rad Laboratories (Hercules, CA, U.S.A.).
6
Western Blot Analysis of REV-ERBα and REV-ERBβ
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Muscle lysates were prepared in RIPA buffer (Sigma) supplemented with Halt protease and phosphatase inhibitor cocktail (#78446, Thermo Scientific), protein concentration determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA), and SDS-PAGE performed using NuPAGE 4%–12% gels (Invitrogen). Proteins were transferred onto a PVDF membrane and incubated with rabbit anti-REV-ERBα (kind gift from Ron Evans), mouse anti-REV-ERBβ (D-8; sc-398252, Santa Cruz), or mouse anti-GAPDH (ab8245, Abcam). Membranes were then incubated with HRP-conjugated donkey anti-rabbit (sc-2317, Santa Cruz) or goat anti-mouse (170–6516, Biorad) secondary antibodies. HRP activity was measured by chemiluminescence (Immobilon Western, Millipore), and bands visualized on CP-BU Medical X-Ray Film (Agfa HealthCare NV, Gevaert, Belgium).
7
Measuring 14-3-3 Degradation in Cells
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Lysates of SHSY5Y cells or zebrafish brain tissue (5 mg protein) were separated in 10% TGX gels (Bio-Rad) and transferred onto PVDF membrane (Bio-Rad) using the Transblot system (Bio-Rad). Western blotting membranes were incubated with rabbit anti-pan-14-3-3 (sc-629, RRID: AB_2273154; 1:1000; Santa Cruz Biotechnology) as primary antibody. Mouse anti-b-actin (A1978, RRID: AB_476692; 1:1000; Sigma-Aldrich) or rabbit anti-GAPDH (ab9485, RRID: AB_307275;1:1000; Abcam) was used as loading control, and goat anti-rabbit (170-6515, RRID: AB_11125142; 1:1000; Bio-Rad) and goat anti-mouse (170-6516, RRID: AB_11125547; 1:1000; Bio-Rad) were used as secondary antibodies. Ebselen-treated samples were referenced to untreated, which were given the arbitrary value of 1.
For experiments on 14-3-3 degradation in cells, 500,000 SH-SY5Y cells per well were seeded in six-well plates; 5 hours postseeding, cells were treated with 5-10 mM ebselen or ebselen oxide and 50 mM Z-VAD-FMK (caspase inhibitor; Adooq Biosciences) and DMSO (0.05%) as a control (without ebselen and caspase inhibitor). Cells were incubated for 16 hours before treating with 500 nM bortezomib (proteasome inhibitor; Fisher Scientific) for 2 hours before collecting for Western blot analysis. Densitometric analysis was performed using Image Laboratory Software 6.0.1 (Bio-Rad).
For experiments on 14-3-3 degradation in cells, 500,000 SH-SY5Y cells per well were seeded in six-well plates; 5 hours postseeding, cells were treated with 5-10 mM ebselen or ebselen oxide and 50 mM Z-VAD-FMK (caspase inhibitor; Adooq Biosciences) and DMSO (0.05%) as a control (without ebselen and caspase inhibitor). Cells were incubated for 16 hours before treating with 500 nM bortezomib (proteasome inhibitor; Fisher Scientific) for 2 hours before collecting for Western blot analysis. Densitometric analysis was performed using Image Laboratory Software 6.0.1 (Bio-Rad).
8
Western Blot Protein Analysis
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Twenty-five to fifty micrograms of protein lysates were analysed by SDS polyacrylamide gel electrophoresis (PAGE). After transfer to a 0.45 l nitrocellulose membrane (GE Healthcare, Amersham, Thermo Fisher Scientific), visualization of blotted protein with ponceau S (Applichem A2935) and blocking in 10 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.05% (v/v) Tween 20 (TBST) containing 2.5% (w/v) skim milk, specific proteins were identified using primary antibodies for 16-18 h at 4 °C. A secondary antibody [donkey anti-goat (sc-2020; Santa Cruz), goat anti-rabbit (1706515; Bio-Rad, Feldkirchen, Germany) or goat antimouse (1706516; Bio-Rad)] conjugated to horseradish peroxidase, and the luminol enhancer detection system (32106, Pierce, Thermo fisher Scientific) was used to visualize the proteins.
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