Western blotting was conducted as described previously (Morita et al., 2015 (link)). Membranes were probed with the primary antibodies listed in Table S5 . HRP-conjugated anti-rabbit IgG (sc-2004; Santa Cruz Biotechnology), anti-mouse IgG (sc-516102; Santa Cruz Biotechnology) and anti-goat IgG (#31402, Thermo Fisher Scientific) were used as the secondary antibodies. Blots were incubated with SuperSignal West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific) and then visualized with ImageQuant LAS4000 (GE Healthcare Life Sciences).
Anti goat igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-goat IgG is a laboratory reagent that binds to goat immunoglobulin G (IgG) proteins. It is commonly used in various immunological techniques, such as Western blotting, ELISA, and immunoprecipitation, to detect and quantify the presence of goat IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg, by Thermo Fisher Scientific (9 mentions)
Anti mouse igg, by Thermo Fisher Scientific (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Gapdh, by Abcam (2 mentions)
Bcl 2, by Abcam (2 mentions)
Caspase 3, by Abcam (2 mentions)
Anti mouse, by Cell Signaling Technology (2 mentions)
Anti rabbit, by Cell Signaling Technology (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Merck Group (2 mentions)
Goat anti ace2, by R&D Systems (2 mentions)
Anti human cy3, by Jackson ImmunoResearch (2 mentions)
Mouse anti ace2, by Abcam (2 mentions)
Anti mouse igg alexa fluor 488, by Jackson ImmunoResearch (2 mentions)
Zen live imaging time lapse microscope, by Zeiss (2 mentions)
8 well chamber slide, by Thermo Fisher Scientific (2 mentions)
40 μm strainer, by BD (2 mentions)
Arginase 1, by Santa Cruz Biotechnology (2 mentions)
Tissue lyser, by Miltenyi Biotec (2 mentions)
Facsverse, by BD (2 mentions)
27 protocols using anti goat igg
1
Western Blotting Technique and Analysis
2
Western Blot Analysis of Osteogenic Markers
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The whole-cell lysate was obtained according to the protocol mentioned above. The lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Boston, MA, USA). After blocking with 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBS-T), the membranes were incubated with primary antibodies. The primary antibody for collagen I (Col I) was purchased from Abcam. The antibody for osteopontin (OPN) was purchased from Novus. The antibodies for dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and runt-related transcription factor 2 (RUNX2) were purchased from Thermo Fisher Scientific. The antibodies for lysosomal-associated membrane protein 1 (LAMP1), β-Actin, ERK, phospho-ERK (p-ERK), P38, phospho-P38 (p-P38) were purchased from Cell Signaling Technology. The antibody for glyceraldehyde-phosphate dehydrogenase (GAPDH) was purchased from Santa Cruz Biotechnology. The secondary antibodies were anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG (Thermo Fisher Scientific, IL, USA).
3
Intestinal Epithelial Cell Phenotyping
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Colon cells were stained with Fc Block (Clone 2.4G2) followed by staining with fluorescently labelled antibodies in IEC buffer (PBS with 5% FBS and 2 mM EDTA to reduce cell clumping) for IECs or 2% FBS in PBS for T cells on ice in 1.5 ml microcentrifuge tubes. For intracellular staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm kit (BD Bioscience). Samples were acquired on an Attune NxT flow cytometer (Life Technologies) and analysed with FlowJo software. Cells were sorted on either a BD ARIA II or S6 Enceladus (BD Biosciences). The following antibodies/reagents were used: anti-CD45 (30-F11; Biolegend), anti-EPCAM1 (G8.8; ThermoFisher), anti-FABP2 (polyclonal; R&D/Fisher), anti-goat IgG (ThermoFisher), anti-LY6G (1A8; BioLegend), anti-MHCII (M5/114.15.2; Fisher), Live/Dead Fixable Near-IR dead cell dye (ThermoFisher), anti-CD4 (RM4-5; BioLegend), anti-TCRβ (Η57−597; ThermoFisher), anti-CD44 (IM7; BioLegend), anti-CD45.1 (A20; ThermoFisher), anti-CD45.2 (104; BD Biosciences), anti-IL-17A (TC11-18H10; BD Biosciences), anti-IFNγ (XMG1.2; ThermoFisher), and anti-IL-22 (1H8PWSR; ThermoFisher).
4
Western Blot Protein Quantification
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For Western blot, total protein was obtained from tissues and DEFs using lysis buffer (Beyotime, Shanghai, China) following the manufacturer's instructions. Whole-cell extracts (30 μg) were separated by SDS-PAGE, transferred onto a polyvinylidene difluoride membrane (Millipore, Danvers, MA), blocked with 5% nonfat milk in TBS-Tween-20 (10-mmol/L Tris-HCl, pH 7.5, 150-mmol/L NaCl, 0.1% Tween-20), and incubated overnight at 4°C with specific primary antibodies against Bcl-2 (1:500; Abcam, Cambridge, UK), caspase 3 (1:1000; Abcam), TNF-α (1:500; Abcam), iNOS (1:1000; Abcam), COX2 (1:500; Bioybyt, Shanghai, China), and GAPDH (1:10000; Abcam). Then membranes were incubated for 1 h with appropriate horseradish peroxidase-conjugated secondary antimouse IgG, antirabbit IgG, and antigoat IgG (1:5000; Thermo Fisher Scientific, Bohemia, NY). Protein levels were quantified using the Image J software.
5
Antibody Validation and Dilution Protocol
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Antibodies were commercially obtained, validated and used according to the manufacturer’s instructions. Primary antibodies, their host species, clonality and dilution are listed in Table S1 . Horseradish peroxidase-labeled secondary antibodies were anti-rabbit (Cat. # 7074, RRID: AB_2099233, Cell Signaling Technology), anti-mouse (Cat# 7076, RRID: AB_330924, Cell Signaling Technology), and anti-goat IgG (Cat# A24452, RRID: AB_2535921, Thermo Fisher Scientific, Waltham, MA, USA).
6
Antibody Detection Protocol
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Antibodies were commercially obtained and used according to the manufacturer’s instructions. Primary antibodies are listed in Table S1 . Horseradish peroxidase-labeled secondary antibodies were anti-rabbit (Cell Signaling Technology, 3 Trask Ln, Danvers, MA, USA, #7074), anti-mouse (Cell Signaling Technology #7076), and anti-goat IgG (Thermo Fisher Scientific #A24452).
7
Western Blot Analysis of Spleen Proteins
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Proteins in the spleen tissues were obtained as described in the lysis buffer (Beyotime, Shanghai, China), following the manufacturer's instructions. The protein concentrations were measured using the BCA Protein Assay Kit (Beyotime), and all samples in the experiment were normalized to equal protein concentrations. Protein samples were separated by SDS-PAGE, transferred onto a PVDF membrane (Millipore, Billerica, MA), blocked with 5% non-fat milk in TBS-Tween-20 (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20), and incubated with specific primary antibodies against Bcl-2 (1:500; Abcam, Cambridge, UK), caspase 3 (1:1,000; Abcam), iNOS (1:1,000; Abcam), COX2 (1:500; Bioybyt, Shanghai, China), and GAPDH (1:10,000; Abcam) overnight at 4°C. The membranes were incubated for 1 h with appropriate HRP-conjugated secondary anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG (1:5,000; Thermo Fisher Scientific), followed by enhanced chemiluminescence (ECL ) detection. The results were normalized to GAPDH as an internal control. Protein levels were quantified using the ImageJ software.
8
Western Blot Analysis of Cellular Proteins
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I7nuc- and pLNCX- infected SiHa cells were lysed with cold high salt extraction buffer, (50 mM Tris-HCl pH 7.5, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.5% NP-40 and 10% glycerol supplemented with 20 mM beta-glycerophosphate, 1 mM PNPP, 1 mM Na3VO4, 1 mM PMSF, 2 mM dithiothreitol and PIC; Sigma-Aldrich). Whole cell extracts (20 μg) were separated by 12% SDS-PAGE, and blotted to PVDF membrane (Immobilon P, Millipore, Billerica MA, USA). Unspecific binding sites were blocked by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% nonfat milk, followed by overnight incubation at 4°C with primary antibodies specific for anti-p53 (DO-1, Novocastra, UK), anti-β-actin (Santa Cruz biotechnology) or anti-V5 tag mAb (Life Technologies), and decoration by peroxidase-labeled anti-mouse IgG (Sigma-Aldrich), or anti-goat IgG (Thermo scientific, Rockford IL, USA), (Sigma-Aldrich), and chemiluminescence detection (Luminata Crescendo Western HRP substrate, Millipore, Billerica MA, USA). Quantitative evaluation of proteins was determined by BioSpectrum Imaging system analysis using the VisioWorksLs software program (UVP, CA, USA).
9
RSV F Protein Detection by Western Blot
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HEp-2 cells were infected with RSV at an MOI of 0.1, incubated for 72 h, harvested and lysed with EzApply (Atto, Tokyo, Japan), and boiled at 100 °C for 5 min. Proteins were separated on 4–12% NuPAGE Bis–Tris gels (Invitrogen Co., Carlsbad, CA, USA), transferred to a polyvinylidene difluoride membrane (Invitrogen), blocked with 4% skim milk solution at room temperature for 1 h, rinsed with 1% skim milk solution. The membrane was probed with anti-RSV F protein antibody, Palivizumab (1:1000) (AstraZeneka, Cambridge, UK), or anti-RSV (AB1128) goat polyclonal antibody (1:500) (Sigma-Aldrich) followed by HRP-conjugated anti-human IgG (1:5000) (Thermo Fisher Scientific) or anti-goat IgG (1:10,000) (Thermo Fisher Scientific), and detected using ECL Prime Western Blotting Detection Reagent (Cytiva, Tokyo, Japan). Images were captured using ChemiDoc XRS + (Bio-Rad Laboratories Inc., Hercules, CA, USA) and the intensity of dots was calculated using ImageJ47 .
10
Immunostaining Protocol for Cellular Imaging
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Immunostaining was performed as previously described (Kim et al., 2015 (link)). The primary antibodies used for ICC are listed in Supplementary file 1 . The secondary antibodies were diluted in PBS and applied for 1 hr: Alexa Fluor 488/555/568/594 anti‐mouse IgG, IgG1, IgM, anti-chicken IgY, anti-rabbit IgG, and anti‐goat IgG (Invitrogen, 1:1,000). Nuclei were stained with DAPI (Invitrogen).
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