One hundred micrograms of denatured total proteins from liver, extracted using RIPA buffer (Merck, Germany) were separated by SDS-PAGE electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, MA, USA). Membranes were overnight incubated at 4°C with 1:1000 dilution of phospho-STAT3 (Tyr705, #9138 Cell Signaling, Beverly, MA, USA), STAT3 (#4904 Cell Signaling, Beverly, MA, USA) and β-actin (#4967 Cell Signaling, Beverly, MA, USA) primary antibodies followed by 1 h incubation at RT with horse anti-mouse IgG-HRP at 1:5000 (#7076 Cell Signaling, Beverly, MA, USA) for phospho-STAT3 antibody binding and goat anti-rabbit IgG-HRP at 1:5000 (#7074 Cell Signaling, Beverly, MA, USA) for STAT3 and β-actin antibodies binding. Chemiluminescence signal was enhanced by adding ECL (SuperSignalTM West Dura Extended Duration Substrate, Thermo Fisher Scientific, MA, USA) and quantified using ImageJ 1.8 software.
Horse anti mouse igg hrp
Manufactured by Cell Signaling Technology
Sourced in United States
Horse anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed for detection and quantification of mouse immunoglobulin G (IgG) in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg hrp, by Cell Signaling Technology (7 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 anti rabbit, by Thermo Fisher Scientific (1 mentions)
P tyr 100, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Vinculin e1e9v, by Cell Signaling Technology (1 mentions)
Donkey anti sheep igg hrp, by Thermo Fisher Scientific (1 mentions)
Ak 15, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Goat anti rabbit igg peroxidase, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
β actin, by GeneTex (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
15 protocols using horse anti mouse igg hrp
1
Western Blot Analysis of Phospho-STAT3
2
Phosphorylation Detection in Cell Biology
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For KiPIK screening, immunoblotting and immunofluorescence microscopy, rabbit polyclonal antibodies to H3T3ph (B8634)47 (link), INCENP (P240, Cell Signaling Technology #2807), INCENP-S446ph (Jan-Michael Peters, IMP, Vienna)57 (link), INCENP-TSSph (Michael Lampson, University of Pennsylvania)82 (link), BCL9L S915ph (Cell Signaling Technology #13325), Neurogranin S36ph (Merck-Millipore, ABN426) and γ-tubulin (AK-15, Sigma, T3320); rabbit monoclonal antibodies to the S-pT-P motif (D73F6; Cell Signaling Technology #5243), Cyclin B1 (D5C10; Cell Signaling Technology #12231) and Vinculin (E1E9V; Cell Signaling Technology #13901); mouse monoclonal antibodies to H3S28ph (CMA315)83 (link) and phospho-tyrosine (P-Tyr-100; Cell Signaling Technology #9411); and sheep polyclonal antibodies to Aurora B (SAB.1, Stephen Taylor, University of Manchester)84 (link) and BCL9L (R&D Systems, AF4967) were used. Antibodies used for siRNA screening were mouse monoclonal anti-H3T3ph (16B2)85 (link) and rabbit polyclonal anti-H2BS6ph86 (link). Secondary antibodies were: donkey anti-sheep IgG-HRP (ThermoFisher, A16041), goat anti-rabbit IgG-HRP (Cell Signaling Technology #7074), horse anti-mouse IgG-HRP (Cell Signaling Technology #7076), donkey anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 594 and anti-sheep Alexa 488 Fluor (ThermoFisher, A-21202, A-21207, and A-11015).
3
Western Blot Analysis of Mitochondrial Proteins
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Cells were sonicated (2 × 15 s at 8% amplitude) in cell lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, and protease inhibitor cocktail (Roche 11836153001). Protein amount was determined using the BCA assay. Protein extracts (15–40 µg) in Laemmli buffer were resolved on a 10% polyacrylamide gel, and transferred to a HybondTM P 0.45 PVDF-membrane (Amersham). Membranes were incubated overnight at 4 °C with the primary antibodies. The primary antibodies used for western blotting were as follows: β-actin (Gene Tex 124214, 1:10,000), MGME1 (Sigma HPA040913, 1:500), POLG (Santa Cruz SC-390634, 1:500), LIG3 (Sigma HPA006723, 1:500), TWNK (Sigma HPA002532, 1:1,000), FEN1 (Sigma HPA006748, 1:250), and ENDOG (Abcam ab9647, 1:1,000). Detection was performed with horseradish peroxidase-conjugated secondary antibodies (horse anti-mouse IgG HRP, Cell Signalling Technology 7076P2, 1:2,000; or goat anti-rabbit IgG–Peroxidase, Sigma A0545, 1:20,000) and SuperSignal West Pico Plus chemiluminescent substrate (Thermo Scientific) and signal was recorded on a ChemiDoc Imaging System (Bio-Rad).
4
Western Blot Secondary Antibody Dilution
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All secondary antibodies were diluted to 1:2000. Rabbit: goat anti-rabbit IgG-HRP (Cell Signaling, Danvers, MA, USA). Mouse: horse anti-mouse IgG-HRP Cell Signaling, Danvers, MA, USA).
5
Western Blot Analysis of CYP2A13
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WB analysis was performed according to previously described procedures [40 (link)]. The primary antibodies against CYP2A13 (Invitrogen, PA5-101310) and GAPDH (Cell Signaling Technology, 97166S) were diluted at a ratio of 1:1000. The secondary antibodies, Goat anti-rabbit IgG-HRP (Cell Signaling Technology, 7074S,) and Horse anti-mouse IgG-HRP (Cell Signaling Technology, 7076S), were diluted at a ratio of 1:3000. The reference protein GAPDH was used to calibrate the errors generated by sample loading.
6
Western Blot Analysis of EMT Markers
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Cell lysates were sonicated in radioimmunoprecipitation assay (RIPA) lysis buffer (sc-24948A, Santa Cruz Biotechnology, Dallas, TX). Protein concentrations were measured with the DC Protein Assay (5000112, Bio-Rad) and RIPA buffer was used as the blank standard. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (10600023, GE Healthcare, Chicago, IL). Following the transfer, PVDF membranes were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and probed with primary antibodies to SNAI1 (1:1000, 3879 S, Cell Signaling Technology), EPAS1 (1:1000, 66731-1-Ig, Proteintech), TFAP2C (1:750, 6E4/4, sc-12762, Santa Cruz Biotechnology), and glyeraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, AM4300, Thermo-Fisher) overnight at 4 °C. PVDF membranes were washed three times for 5 min each with TBST and incubated with secondary antibodies (goat anti-rabbit IgG HRP, 1:5000, 7074S, Cell Signaling Technology and horse anti-mouse IgG HRP, 7076, Cell Signaling Technology) for 1 h at room temperature. Immunoreactive proteins were visualized using the LuminataTM Crescendo Western HRP Substrate (WBLUR0500, Millipore, Billerica, MA) according to the manufacturer’s instructions. Full scan blots are presented in the Source Data file.
7
Optimized Antibody Detection Dilutions
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Rabbit: Goat anti-rabbit IgG-HRP, diluted 1:2000 (Cell Signaling), Rat: Goat anti-rat IgG conjugated with HRP, diluted 1:100,000 (Pierce), Mouse: Horse anti-mouse IgG-HRP diluted 1:2000 (Cell Signaling).
8
Western Blot Analysis of Lig3 Protein
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The protein samples extracted from RVLM tissues or B104 cells were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad, USA). The membranes containing proteins were blocked with QuickBlock buffer (Beyotime, China) for 1 h at room temperature. Afterward, the membranes were blotted with rabbit polyclonal Lig3 (1:1000, ABclonal, China) and mouse monoclonal horseradish peroxidase (HRP)-conjugated GAPDH (1:5000, Proteintech, USA) primary antibodies at 4 °C overnight. After washing the membranes three times with PBST, they were hybridized with goat anti-rabbit IgG-HRP (1:3000, Cell Signaling Technology, USA) and horse anti-mouse IgG-HRP (1:3000, Cell Signaling Technology, USA) secondary antibodies for 1 h at room temperature. The signals of proteins were visualized using super ECL detection reagent (Yeasen, China) with the Tanon-5200 automatic chemiluminescence image analysis system (Tanon Science & Technology, China). GAPDH acted as a reference.
9
Regulatory T Cell and Th17 Cell Staining
10
Western Blot Analysis of Retinal Cell Proteins
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Mice retina, RPE, and choroid were isolated via sonication in lysis buffer (iNtRON Biotechnology, Seongnam, Korea) and centrifuged at 13,000 rpm for 25 min at 4 °C. The supernatant was obtained as total protein extracts. ARPE-19 cells were homogenised with lysis buffer (iNtRON Biotechnology) and centrifuged at 13,000 rpm for 25 min at 4 °C. The supernatant was obtained as total protein extracts. An equal amount of total protein was resolved on 12% sodium dodecyl sulfate–polyacrylamide gel followed by gel electrophoresis. The transferred PVDF membranes were incubated overnight at 4 °C with alpha-SMA (Abcam, ab5694, 1:1000), VEGFA (Abcam, ab46154, 1:1000) CRYAB (Abcam, ab13496, 1:2500), phospho-CRYAB (Ser59; Invitrogen, PA1-012, 1:2500), and GAPDH (Cell Signaling Technology, 2118S, 1:5000) antibodies. Thereafter, blots were incubated with corresponding conjugated goat anti-rabbit (Cell Signaling, 7074S, 1:5000) or horse anti-mouse IgG-HRP (Cell Signaling, 7076S, 1:5000) secondary antibodies. Immunoreactive proteins were detected with ECL substrate (Millipore) and visualised using a FUSION Solo S chemiluminescence detection system (Vilber Lourmat, Collégien, France).
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