Sample preparation for western blotting was conducted as described44 (link). Briefly, denuded oocytes (n = 50) were collected and frozen in Laemmli buffer with protease inhibitors (Sigma). Prior to gel electrophoresis, samples were boiled at 100 °C for 5 min and then separated in 7.5% acrylamide gels containing 0.1% SDS. Proteins were transferred onto hydrophobic PVDF membranes (Amersham). Membranes were blocked with 5% non-fat milk in PBST (PBS with 0.1% Tween-20) overnight at 4 °C and then incubated with rabbit anti-LSH antibody (a gift from Dr. Kathrin Muegge, National Cancer Institute, 1/5000 in PBST) or anti-H3T3ph (EMD Millipore, 1:1000 dilution) at 4 °C overnight and subsequently washed in PBST. After three washes in PBST for a total of 60 min, the membranes were labeled with peroxidase-conjugated goat anti-rabbit IgG (Invitrogen, 1/5000 in PBST) for 1 h. The proteins were visualized using an ECL-plus detection system (Amersham). Some membranes were also probed with anti-β tubulin (Sigma Aldrich, 1/2000 dilution) as an internal control. Uncropped/unprocessed scans of all western blots can be found in the Source Data file.
Peroxidase conjugated goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Peroxidase-conjugated goat anti-rabbit IgG is a secondary antibody used in various immunoassay techniques. It binds to rabbit primary antibodies and is conjugated with the enzyme horseradish peroxidase, which can be used for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Ecl plus detection system, by Cytiva (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
Amersham hybond ecl, by GE Healthcare (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Hybond, by Cytiva (1 mentions)
Anti bax pab, by Santa Cruz Biotechnology (1 mentions)
Anti β actin mab, by Merck Group (1 mentions)
Toto 3, by Thermo Fisher Scientific (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
P8340, by Merck Group (1 mentions)
P5726, by Merck Group (1 mentions)
Blocking one p, by Nacalai Tesque (1 mentions)
Rabbit anti total erk1 2, by Cell Signaling Technology (1 mentions)
Las 4000 luminescent image analyzer, by Fujifilm (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
P2850, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
13 protocols using peroxidase conjugated goat anti rabbit igg
1
Western Blot Protocol for Oocyte Protein Analysis
2
Protein Expression Analysis by Western Blot
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Protein samples were boiled for 5 min in Laemmli loading buffer, size-fractionated by SDS/PAGE and transferred to Amersham Hybond-ECL (GE Healthcare, RPN2020D) membranes. After blocking with 5% milk in PBS for at least 1 h at room temperature or overnight at 4°C under agitation, membranes were incubated with antibodies for 1 h at room temperature or overnight at 4°C under agitation in 1% milk in PBS. The primary antibodies, all raised in rabbits, were used at the following dilutions: ATG24 (1:4,000); RAB11 (1:2,000, gift from Prof. M. Field, Dundee); ZFP3 (1:10,000, gift from Prof. K. Matthews, Edinburgh); PPDK (gift from Dr F. Bringaud, Bordeaux; 1:25,000). As secondary antibody, peroxidase-conjugated goat anti-rabbit IgG; Invitrogen) was used at 1:10,000. After extensive washing in PBS then 0.05% NP40 in PBS, blots were revealed using Pierce ECL western blotting substrate (Thermo Scientific, 32106).
3
Apoptosis Induction Assay Protocol
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N-(3-oxododecanoyl)-homoserine lactone (C12), polybrene and actinomycin D were purchased from Sigma (St. Louis, MO). Propidium iodide (PI), CM-H2DCFDA (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester), TMRE (tetramethylrhodamine, ethyl ester) and TOTO-3 were obtained from Invitrogen (Carlsbad, CA). Unless otherwise stated, all reagents were dissolved in dimethyl sulfoxide (DMSO). Dulbecco's Modified Eagle's Medium (DMEM), penicillin/streptomycin, trypsin, and L-glutamine were obtained from Mediatech (Manassas, VA), and fetal bovine serum was purchased from Gemini (Broderick, CA). Caspase-Glo assay 3/7 kit was purchased from Promega (Madison, WI). Antibodies (Abs) for western blot were anti-β-actin mAb (Sigma), anti-Bcl-2 mAb; anti-Bax pAb (Santa Cruz; Dallas, TX), anti-Bak pAb (Millipore; Billerica, MA), anti-PON2 pAb; anti-PON3 pAb; anti-Mcl-1 pAb (Abcam; Cambridge, MA), anti-Noxa pAb (Novus; Littleton, CO), anti-Puma pAb (ProSic; Poway, CA) peroxidase-conjugated goat anti-rabbit IgG (Thermo Fisher; Waltham, MA) and peroxidase-conjugated goat anti-mouse IgG (Thermo Fisher).
4
Immunohistochemical Analysis of Heart Tissue
5
Immunoblotting of Phosphorylated ERK1/2
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STHdh cells were washed with PBS, harvested, and lysed using RIPA buffer (R0278; Sigma) with protease (P8340; Sigma) and phosphatase inhibitor cocktails (P2850 and P5726; Sigma). Lysates were solubilized in sodium dodecyl sulphate (SDS) sample buffer, separated on 10% SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; EMD Millipore Corporation, Billerica, MA). Transfer membranes were blocked for 30 min at room temperature with Blocking One-P (Nakalai Tesque, Kyoto, Japan), then incubated overnight at 4°C with the primary antibodies (rabbit anti-phospho-extracellular signal-regulated kinase 1/2 [ERK1/2] [Cell Signaling Technology, Danvers, MA], rabbit anti-total-ERK1/2 [Cell Signaling Technology], and mouse anti-β-actin [Sigma]). Subsequently, the membranes were incubated with secondary antibodies (peroxidase-conjugated goat anti-rabbit IgG [Thermo Fisher Scientific Inc., Franklin, MA] or peroxidase-conjugated goat anti-mouse antibody [Thermo]). The immunoreactive bands were visualized using ImmnoStar®LD (Wako Pure Chemical Industries, Osaka, Japan) and the LAS-4000 Luminescent Image Analyzer (Fuji Film Co., Ltd., Tokyo, Japan).
6
Quantification of Runx2 Expression in Chondrocytes
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The total protein was extracted from the cultured chondrocytes according to the manufacturer’s protocols using an RIPA buffer (Beyotime, Shanghai, China). After denaturation, 30 μg of total protein was electrophoresed for immunoblot analysis. The blots were probed with primary antibodies directed against runx2 (Abcam, Cambridge, UK) overnight at 4 °C. GAPDH polyclonal antibody (Bioworld, Minneapolis, MN, USA) was used as a housekeeping reference. Peroxidase-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA) or goat anti-mouse IgG (Bioss, Shanghai, China) antibodies were used to visualize proteins using Western blotting chemiluminescence luminol reagent on a GeneGnome XRQ Western Blotting Analysis System (Syngene, Frederick, MD, USA). Working concentrations for each antibody were determined empirically based on the recommended stock solutions. Image J was used to quantify the band intensities of proteins of interest in the experimental and control groups.
7
Quantitative Western Blot Analysis of Tight Junction Proteins
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Western blot was performed as we previously described5 (link). Equal amounts of protein per sample were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Blots were incubated overnight with primary antibodies: rabbit anti-CLDN1 (1:500; Abcam, Cambridge, UK), rabbit anti-CLDN2 (1:500; Abcam), rabbit anti-CLDN3 (1:500; Abcam), mouse anti-CLDN4 (1:1000; Invitrogen, Carlsbad, USA), rabbit anti-OCLN (1:1000; Invitrogen), rabbit anti-DSC2 (1:500; Abcam), mouse anti-DSG2 (1:1000; Abcam), mouse anti-tryptase (1:500; Dako, Glostrup, Denmark) or mouse anti-MBP (1:500; AbD Serotec, Kidlington, UK). All membranes were stained with mouse anti-vinculin (1:5000; Sigma-Aldrich), as a protein loading control. Peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (both 1:5000; Thermo Scientific) were used as secondary antibodies. Bands were quantified by densitometry using ImageJ software (National Institutes of Health; https://rsb.info.nih.gov.ij/ ). Fold change was determined relative to the average of the group perfused with the saline solution (part 1) or the placebo oral treatment (part 2).
8
Recombinant REV1 Protein Expression and Purification
9
Alendronate Modulates Cell Signaling in Osteoblasts
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Nitrogen-containing BP, alendronate sodium salt trihydrate (FOS) was purchased from Wako in Japan. SCGMTM BulletKitTM, N-acetyl-cysteine (NAC), fluorescein isothiocyanate (FITC)-conjugated annexin V, and ECL chemiluminescence were acquired from Sigma in China. PTHrP, cell-permeable 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), U0126 (inhibitor of ERK1/2), SB203580 (inhibitor of p38), sanguinarine chloride (inhibitor of MKP1), lipofectamine LTX transfection reagent, Tris HCl, NaCl, EDTA, 1% Triton X-100, 1% sodium hydroxide, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail, cocktail Set II, bovine serum albumin (BSA), Considering bicinchoninic acid (BCA), nitrocellulose membrane were obtained from ThermoFisher in China. In addition, α-Tubulin, peroxidase-conjugated goat anti-rabbit IgG and the antibodies of p-ERK1/2, ERK1/2, p-Akt, Akt, p-p38, p38, p-JNK, JNK, MKP-1, and p-p66Shc (Ser36) were acquired from ThermoFisher in China. Propidium iodide was purchased from Solarbio in China.
10
Mammalian Cell Culture Protocol
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Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin/streptomycin, trypsin, and L-glutamine were obtained from Mediatech (Manassas, VA). Fetal bovine serum (FBS) was purchased from Gemini (Broderick, CA). Polybrene, N-(3-oxododecanoyl)-homoserine lactone (C12), puromycin, [U-13C]-glucose, deuterium oxide (D2O), 2-Dimethyl-2-silapentane-5-sulfonate sodium salt (DSS), collagenase, and heparin were purchased from Sigma-Aldrich (St. Louis, MO). Lipofectamine2000® transfection reagents, propidium iodide, β-Mercaptoethanol and Matrigel® were purchased from Thermo Fisher (Waltham, MA). Antibodies (Abs) for western blot were anti-β-actin mAb (A5441; Millipore Sigma; Burlington, MA), anti-Mouse PON2 pAb (ABIN1573944; antibodies-online.com; Atlanta, GA), peroxidase-conjugated goat anti-rabbit IgG (65-6120; Thermo Fisher; Waltham, MA) and peroxidase-conjugated goat anti-mouse IgG (65-6520; Thermo Fisher). Ad5-CMV-Cre was purchased from the Baylor College of Medicine Gene Vector Core.
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