Total protein from each sample was extracted using the method described by Zhang et al (6 (link)). Proteins (40 µg) were separated on 10% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% milk at 25°C for 2 h and membranes incubated with 1:2,000 diluted rabbit anti-phosphorylated (p)-SMAD2/3 (sc-517575; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), NOX2 (sc-130543; Santa Cruz Biotechnology, Inc.), NOX4 (sc-30141; Santa Cruz Biotechnology, Inc.), ALK5 (sc-101574; Santa Cruz Biotechnology, Inc.), caspase-3 (sc-7272; Santa Cruz Biotechnology, Inc.) and mouse anti-β-actin (1:2,000; sc-47778; Santa Cruz Biotechnology, Inc.) at 4°C for 16 h followed by incubation with HRP-goat anti-mouse IgG (1:2,000; A0216; Beyotime Institute of Biotechnology) or anti-rabbit IgG (1:2,000; A0208; Beyotime Institute of Biotechnology). Bands were detected using an enhanced chemiluminescence kit (GE Healthcare, Chicago, IL, USA) and a Molecular Imager ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometric analysis was conducted with ImageJ 1.43 (National Institutes of Health, Bethesda, MD, USA).
Goat anti mouse igg hrp
Manufactured by Beyotime
Sourced in China
Goat anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used to detect and quantify mouse immunoglobulin G (IgG) in various immunoassays, such as ELISA, Western blotting, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg hrp, by Beyotime (11 mentions)
Chemidoc imaging system, by Bio-Rad (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Trans blot electrophoretic transfer kit, by Bio-Rad (2 mentions)
Cell lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Rabbit anti il 1β, by Abcam (2 mentions)
Rabbit anti tnf α, by Abcam (2 mentions)
Rabbit polyclonal anti cyr61, by Thermo Fisher Scientific (2 mentions)
Donkey anti goat igg hrp, by Beyotime (2 mentions)
Mouse anti il 6, by Abcam (2 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
Caspase 3, by Santa Cruz Biotechnology (1 mentions)
Ha c5, by Abcam (1 mentions)
Beyoecl plus, by Beyotime (1 mentions)
19 protocols using goat anti mouse igg hrp
1
Western Blot Analysis of Protein Signaling
2
Western Blot Analysis of Protein Expression
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Total cellular protein was extracted using the cell lysis buffer (Beyotime), and concentrations were determined by BCA protein assay kit (Beyotime). Lysates were loaded in SDS-AGE gel and electrophoresed. Proteins were transferred from gel to nitrocellulose membrane using a trans-blot electrophoretic transfer kit (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked in 5% skim milk in TBST buffer and incubated with primary antibodies anti-JMJD6 (1:3000; H-7, Santa Cruz Biotechnologies, Dallas, TX, USA), anti-histone H4R3me2a (1:3000; active motif), anti-HA tag (1:4000; HA.C5, Abcam), anti-α-tubulin (1:6000, DM1A, CST), anti-PDE1C (1:2000; Abcam) and anti-GAPDH (1:4000; 6C5, Abcam). After washing, the membranes were incubated with HRP goat anti-mouse IgG (Beyotime) or HRP goat anti-rabbit IgG (Beyotime). Membranes were then incubated in BeyoECL plus (Beyotime) and then imaged using Chemidoc imaging system (Bio-Rad Laboratories). Gray value was analyzed using Image J (Java 1.8.0).
3
Apoptosis Mechanisms in Tca8113 Tongue Cancer
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AA was obtained from the National Institute for Food and Drug Control (Nanjing Jingzhu, China). Antibodies specific for calpain, cleaved caspase-3, JNK, p-JNK, Grp78, Bax, and Bcl-2, and actin were from Cell Signaling Technology (Shanghai, China), while Beyotime Biotechnology (Shanghai, China) was the source of BCA kits, Fluo-4AM fluorescent dye, SDS-PAGE gel preparation kits, secondary HRP goat anti-rabbit IgG, HRP-goat anti-mouse IgG, anti-IRE1α, and anti-P-IRE1α. The human Tca8113 tongue cancer cell line was obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China).
4
Osteogenic Differentiation Protein Analysis
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After 21 days osteogenic differentiation of AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs, the total cellular protein was extracted using the cell lysis buffer (Beyotime), and concentrations were determined by Bradford protein assay kit (Bio-Rad). Proteins were loaded in SDS-AGE gel and electrophoresed at 80 V for 30 min and 140 V for 60 min. Then, proteins were transferred from gel to nitrocellulose membrane using a trans-blot electrophoretic transfer kit (Bio-Rad). Membranes were blocked in 5% skim milk in TBST buffer for 60 min and incubated with primary antibodies osterix (1:3000, ab94744, abcam 45kd), collagen I (1:3000, ab34710, abcam 125kd), osteopontin (1:2000, ab166709, abcam 65kd), osteocalcin (1:2000, ab93876, abcam 11kd), Tubulin(1:4000, ab4074, abcam 50kd), phosphate-Akt (1:2000, 193H12, Cell signaling technology), Total-Akt (1:2000, C67E7, Cell Signaling Technology), phosphate-Erk1/2 (Thr202/Tyr204, Cell Signaling Technology), total- ERK1/2 (1:2000, Cell Signaling Technology). After washing with TBST buffer, the membranes were incubated with HRP goat anti-mouse IgG (1:3000, Beyotime) or HRP goat anti-rabbit IgG (1:3000, Beyotime). Membranes were then incubated with pierce ECL western blotting substrate (Thermo fisher) and then imaged using chemidoc imaging system (Bio-Rad).
5
Autophagy Modulation Assay Protocol
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Ferrozine, neocuproine, ascorbic acid and 3-methyladenine (3-MA) were bought from Sigma-Aldrich (USA). Ammonium acetate and 30% hydrogen peroxide (H2O2) solution were purchased from Aladdin (China). Antibody against LC3 (NB100-2220) and P62 (ab56416) were purchased from Novus (USA) and Abcam (USA), respectively. PathScan® Intracellular Signaling Array Kit (7323), antibody against PARP (9542T) and Beclin-1 (3495T) and goat-anti-rabbit-IgG-HRP were all obtained from Cell Signaling Technology (USA). Goat-anti-mouse-IgG-HRP was obtained from Beyotime Biotechnology (China). Antibody against β-actin (abs-118937) was bought from Absin (China).
6
Quantifying DNA Methylation Changes
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A total of 2 µl genomic DNA was extracted from tissues (Qiagen GmbH) and was dropped onto each nitrocellulose membrane at a 2-fold serial dilution (0, 5, 10, 20 and 40 ng) for dot blot assay. The spots were dried at room temperature and incubated in TBST with antibodies against 5hmC (cat. no. ab214728; Abcam) or 5-methylcytosine (5mC; cat. no. ab10805; Abcam) (1:500 dilution; 1 ng/ml) in 10 ml of TBS-T for 4 h overnight with gentle shaking. The membranes were washed with TBS-T three times for 10 min each time at room temperature, followed by goat anti-mouse IgG-HRP (1:10,000 dilution; 20 ng/ml; A0216, Beyotime Institute of Biotechnology) or goat anti-rabbit IgG-HRP (1:10,000 dilution; 20 ng/ml; A0208, Beyotime Institute of Biotechnology) incubation in 10 ml of TBST for 1 h at room temperature with gentle shaking, and washed again three times. BSA (New England Biolabs Inc.) was used as a negative control. Membranes were subsequently incubated with 3 ml of ECL Western Blotting Substrate (Beyotime Institute of Biotechnology) for 5 min in darkness at room temperature to develop the bands by Tanon 5200 Chemiluminescence Imaging Analysis System.
7
Protein Expression Analysis in Stem Cells
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Unless otherwise indicated, reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit anti-SHP-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and a mouse anti-Nanog polyclonal antibody was obtained from Bethyl Laboratories (Montgomery, TX, USA). Rabbit anti-STAT3 antibody was purchased from Merck Millipore (Millipore Chemicals, MA, USA) and a rabbit anti-phospho-STAT3 antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-GAPDH polyclonal antibody was purchased from beyotime (Beyotime, Shanghai, China). Secondary antibodies for ICC were alexa fluor 488-labeled goat anti-Rabbit IgG and cy3-labeled goat anti-mouse IgG purchased from beyotime (Beyotime, Shanghai, China). Secondary antibodies for Western were goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP purchased from beyotime (Beyotime, Shanghai, China).
8
Neural stem cell culture protocol
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PQ was purchased from Sigma Chemical Co. (Sigma-Aldrich, Milan, Italy). ReNcell NSC Maintenance Medium and accutase were obtained commercially from Millipore (Temecula, CA). Epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2) were purchased from PeproTech. Laminin was purchased from Invitrogen (Carlsbad, CA, USA). Catalase Assay Kit, Malondialdehyde Assay Kit, Lactate Dehydrogenase Assay Kit, BCA Protein Assay Kit, Cell Lysis Buffer for Western and IP, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP were obtained from Beyotime (Jiangsu, China). Total Superoxide Dismutase Assay Kit was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Tripure was obtained from Roche (Basel, Switzerland). The AMV first strand cDNA Synthesis Kit was purchased from MBI (Fermentas, Canada). Real-time PCR Kit was obtained from Tiangen Biotech (Beijing, China). Rabbit anti-Nrf2 polyclonal antibody, rabbit anti-Keap1 polyclonal antibody, rabbit anti-PKC polyclonal antibody, and rabbit anti-CKII polyclonal antibody were purchased from GeneTex (San Antonio, USA). Mouse anti-β-tubulin polyclonal antibody was purchased from Boster (Wuhan, China).
9
Resveratrol and Nicotinamide Modulate Chondrocyte Autophagy
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Chemicals and antibodies were purchased as follows: resveratrol (Sigma, R5010) dissolved in 100% dimethyl sulfoxide (DMSO; Sigma, D2650) to a concentration of 40 mM, nicotinamide (Sigma, 72340) dissolved in phosphate-buffered saline (PBS) to a concentration of 200 mM, bafilomycin A (Sigma, B1793) dissolved in DMSO to a concentration of 5 μM, 0.25% trypsin solution (Sigma, 59429C), 0.2% type II collagenase (Sigma, V900892), Dulbecco's modified Eagle's medium and Ham's F-12 medium (DMEM/F12, 1:1; Gibco, 11320-033), fetal bovine serum (FBS; Gibco, 10099-141), penicillin-streptomycin (Sigma, G4664), rabbit anti-SIRT1 (Epitomics, ab32441), rabbit anti-Caspase3 (Epitomics, ab32351), rabbit anti-Collagen II (Abcam, ab34712), rabbit anti-LC3 (Cell Signaling Technology, 4599), mouse anti-Beclin-1 (Abcam, ab114071), mouse anti-β-actin (Beyotime, AA128), goat anti-rabbit IgG-HRP (Beyotime, A0208), goat anti-mouse IgG-HRP (Beyotime, A0216).
10
Western Blot Analysis of Innate Immunity Proteins
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Briefly, cells and tissue were lysed with RIPA buffer (Beyotime Biotechnology) supplemented with PMSF (Beyotime Biotechnology) and protease inhibitor cocktail (Roche). METTL3 (abcam, ab195352, 1:2000), METTL14 (sigma, HPA038002, 1:2000), ALKBH5 (sigma, HPA007196, 1:2000), FTO (abcam, ab92821), NSP1 and VP6 (gift from Harry B. Greenberg lab), GAPDH (proteintech), TUBULIN (proteintech), beta-ACTIN (proteintech), Phospho-IRF-7 (Ser437/438) (D6M2I) (CST), Phospho-TBK1/NAK (Ser172) (D52C2) (CST), TBK1/NAK (D1B4) (CST), and IRF-7 (D8V1J) (CST) antibodies were used in accordance with the manufacturer’s instructions. After incubation with the primary antibody overnight, the blotted PVDF membranes (Immobilon, IPVH00010) were incubated with goat anti-rabbit IgG-HRP (Beyotime, A0208) or goat anti-mouse IgG-HRP (Beyotime, A0216) and exposed with BIO-RAD ChemiDocTM Imaging System for a proper exposure period.
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