The tissue was homogenized in RIPA buffer containing a protease inhibitor cocktail (Beyotime Biotechnology). A BCA protein assay (Beyotime Biotechnology) was used to determine the protein concentrations. Equal amounts of protein were separated by SDS-PAGE and then transferred electrophoretically to polyvinylidene fluoride membranes (Bio-Rad) [29 (link), 30 (link)]. The membranes were incubated with the following primary antibodies for 2 h at room temperature: IL-6 (1:1000, Abcam, England), Gp130 (1:1000, Santa, America), pSTAT3 (1:1000, Bioss, China), and NMDA receptors (1:1000, Bioss, China). The membranes were then incubated with GAPDH (1:1000, Solarbio, China), goat anti-mouse IgG (Bioss, China, Gp130, 1:1000 and GAD67, 1:1000) or goat anti-rabbit IgG (Bioss, China, pSTAT3, 1:1000, NMDA receptors, 1:3000 and IL-6, 1:1000) secondary antibodies for 2 h at room temperature. Finally, the membranes were placed in a gel imaging analysis system for exposure and analysis (AlphaView FluorChem FC3).
Goat anti mouse igg
Manufactured by Bioss Antibodies
Sourced in China
Goat anti-mouse IgG is a secondary antibody reagent used to detect and quantify the presence of mouse immunoglobulin G (IgG) in various immunoassays and research applications. It is produced by immunizing goats with mouse IgG and purifying the resulting antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Bioss Antibodies (4 mentions)
Gapdh, by Solarbio (3 mentions)
Gp130, by Bioss Antibodies (3 mentions)
Nmda receptors, by Bioss Antibodies (3 mentions)
Gad67, by Bioss Antibodies (3 mentions)
Pstat3, by Bioss Antibodies (3 mentions)
Interleukin 6 (il 6), by Bioss Antibodies (3 mentions)
Interleukin 6 (il 6), by Abcam (3 mentions)
Bca protein assay, by Beyotime (3 mentions)
Protease inhibitor cocktail, by Beyotime (3 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Gapdh polyclonal antibody, by Bioworld Technology (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Polyvinylidene uoride membrane, by Bio-Rad (1 mentions)
Secondary antibody dilution buffer, by Beyotime (1 mentions)
Primary antibody dilution buffer, by Beyotime (1 mentions)
Rabbit anti camkii, by Abcam (1 mentions)
Rabbit anti glur1, by Abcam (1 mentions)
Rabbit anti nmdar2b, by Abcam (1 mentions)
10 protocols using goat anti mouse igg
1
Protein Expression Analysis in Biological Samples
2
Lateral Flow Assay for Cronobacter spp. Detection
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The composition of LFA which contained five parts (including PVC plate, sample pad, conjugate pad, absorbent pad, nitrocellulose (NC) membrane) obtained from JieYi Biotechnology was demonstrated in Fig. 1 a. The sample pad was saturated with running buffer (100 mM Tris, 0.2% BSA, 0.05% casein, 4% trehalose, 0.5% Tween 20) for 10 min, then dried at 42 °C for 1 h and stored in the refrigerator with desiccants at 4 °C for future use. The SA obtained from Sigma-Aldrich diluted by PB (0.01 M, pH = 7.4) to 2.5 mg/mL and goat anti-mouse IgG (1 mg/mL) purchased from Bioss (Beijing, Chian) were immobilized onto the NC membrane with a volume of 1 μL/cm to form the T and C lines, respectively. Pure cellulose fiber was used as the absorbent pad. The strips were cut into 4 mm width and stored in a desiccator at 4 °C for the detection of Cronobacter spp. ZX1000 Dot Dispensing Plat form XYZ3050 (BioDot, USA) and Guillotine Cutter HGS201 (Autokun, China) were used to dispense reagents and cut the strips, respectively.![]()
Schematic diagram of the lateral flow assay.
3
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.
4
Colloidal Gold Competition Test Strip for MEV
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A nitrocellulose membrane was coated with purified MEV VP2 protein and goat anti-mouse IgG (Bioss, Beijing, China) as the test line (T line) and the control line (C line), respectively. The glass cellulose membrane was used as a sample pad, which was immersed in 0.1 M Tris-buffered saline. The polyester fiber used as the conjugate pad, which was fully immersed in 0.01 M phosphate-buffered saline. Figure 1A shows the principle of the colloidal gold competition test strip for the detection of MEV.
5
Western Blot Analysis of Protein Expression
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The tissue was homogenized in RIPA buffer containing a protease inhibitor cocktail (Beyotime Biotechnology).
A BCA protein assay (Beyotime Biotechnology) was used to determine the protein concentrations. Equal amounts of protein were separated by SDS-PAGE and then transferred electrophoretically to polyvinylidene fluoride membranes (Bio-Rad) [29, 30] . The membranes were incubated with the following primary antibodies for 2 hours at room temperature: IL-6 (1:1000, Abcam, England), Gp130 (1:1000, Santa, America), pSTAT3 (1:1000, Bioss, China), and NMDA receptors (1:1000, Bioss, China). The membranes were then incubated with GAPDH (1:1000, Solarbio, China), goat anti-mouse IgG (Bioss, China, Gp130, 1:1000 and GAD67, 1:1000) or goat anti-rabbit IgG (Bioss, China, pSTAT3, 1:1000, NMDA receptors, 1:3000 and IL-6, 1:1000) secondary antibodies for 2 hours at room temperature. Finally, the membranes were placed in a gel imaging analysis system for exposure and analysis (AlphaView FluorChem FC3).
A BCA protein assay (Beyotime Biotechnology) was used to determine the protein concentrations. Equal amounts of protein were separated by SDS-PAGE and then transferred electrophoretically to polyvinylidene fluoride membranes (Bio-Rad) [29, 30] . The membranes were incubated with the following primary antibodies for 2 hours at room temperature: IL-6 (1:1000, Abcam, England), Gp130 (1:1000, Santa, America), pSTAT3 (1:1000, Bioss, China), and NMDA receptors (1:1000, Bioss, China). The membranes were then incubated with GAPDH (1:1000, Solarbio, China), goat anti-mouse IgG (Bioss, China, Gp130, 1:1000 and GAD67, 1:1000) or goat anti-rabbit IgG (Bioss, China, pSTAT3, 1:1000, NMDA receptors, 1:3000 and IL-6, 1:1000) secondary antibodies for 2 hours at room temperature. Finally, the membranes were placed in a gel imaging analysis system for exposure and analysis (AlphaView FluorChem FC3).
6
Western Blot Analysis of Signaling Proteins
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The tissue was homogenized in RIPA buffer containing a protease inhibitor cocktail (Beyotime Biotechnology). A BCA protein assay (Beyotime Biotechnology) was used to determine the protein concentrations. Equal amounts of protein were separated by SDS-PAGE and then transferred electrophoretically to polyvinylidene uoride membranes (Bio-Rad) [29, 30] . The membranes were incubated with the following primary antibodies for 2 hours at room temperature: IL-6 (1:1000, Abcam, England), Gp130 (1:1000, Santa, America), pSTAT3 (1:1000, Bioss, China), and NMDA receptors (1:1000, Bioss, China). The membranes were then incubated with GAPDH (1:1000, Solarbio, China), goat antimouse IgG (Bioss, China, Gp130, 1:1000 and GAD67, 1:1000) or goat anti-rabbit IgG (Bioss, China, pSTAT3, 1:1000, NMDA receptors, 1:3000 and IL-6, 1:1000) secondary antibodies for 2 hours at room temperature. Finally, the membranes were placed in a gel imaging analysis system for exposure and analysis (AlphaView FluorChem FC3).
7
Immunoblotting of Synaptic Proteins
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The primary antibodies used in the study were the rabbit anti-NMDAR1 (1:5,000), rabbit anti-NMDAR2B (1:1,000), rabbit anti-NMDAR2B (phoshpo-Ser1303) (1:1,000), rabbit anti-GluR1 (1:2,000), rabbit anti GluR1 (phospho-Ser831) (1:1,000), rabbit anti-CaMKII (1:5,000) (Abcam, Cambridge, UK), rabbit anti-CaMKII (phospho-Thr286) (1:1,000) (Cell Signal Technology, Danvers, MA, USA), rabbit anti-beta tubulin (1:1,000) (Proteintech, Wuhan, China) and mouse anti-beta tubulin (1:3,000) (Bioss, Beijing, China). The secondary antibodies were horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (1:10,000) and goat anti-mouse IgG (1:5,000) (Bioss). The antibodies were diluted with the primary antibody dilution buffer or secondary antibody dilution buffer (Beyotime) according to the manufacturer's guidance.
8
Protein Expression Analysis of DMSCs
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DMSCs were rinsed with ice-cold phosphate-buffered saline, and total cell protein extracts were prepared using a cell lysis buffer (Beyotime Technology, Shanghai, China) containing protease inhibitors. Next, 10 mg total protein from the MSCs was boiled with sample loading buffer for 5 min and then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by electro-transfer to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked in 5% skim milk at 4°C overnight and then sequentially incubated with different diluted primary antibodies the next morning [(LITAF): mouse anti-human, 1:2000; (HHEX): rabbit anti-human, 1:2000; (DUSP1): rabbit anti-human, 1:1000; glyceraldehyde 3-phosphate dehydrogenase (GAPDH):mouse anti-human, 1:5000; Abcam, Cambridge, UK] at 4°C overnight followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG; Bioss, Beijing, China) at 37°C for 2 h. Enhanced chemiluminescence was performed for color development and exposure. The ratio in gray scale of the objective band under test to GAPDH was calculated to semi-quantitatively analyze protein levels.
9
Nanomagnetic Immunoassay for PRV Detection
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A combination of 3-mercaptopropionic acid (MPA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and ethanolamine were utilized to chemically modify the surface of SPR chip. Hexaammonium heptamolybdate tetrahydrate ((NH4)6Mo7O24·4H2O) and thiourea (CH4N2S) were used to synthesize MNF. The materials mentioned above were brought from the Shanghai Aladdin Company. Carboxylated NDs with an average size of 35 nm were purchased from the Taiwan FND Biotech Company. PBS was purchased from the Shanghai Sangon Biotech Company. Goat-anti-mouse IgG, mouse IgG, rabbit IgG, and bovine IgG were purchased from the Beijing Biosynthesis Biotechnology Company. PRV, PRV antibody, and pig serum samples were extracted from samples collected on the farm. PRV and PRV antibodies were dissolved in a mixture of pig serum and PBS (3:100 v/v). IgG reagents, PRV, PRV antibody, and serum sample were stored at −20 °C, and other reagents were stored at −4 °C.
10
Western Blot Analysis of TP53 and GAPDH
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Total protein was extracted from cells using a total protein extraction kit according to the manufacturer's instructions (KeyGen, Rockville, MD, USA). The protein concentrations of the lysates were measured with the Protein BCA Assay Kit (Bio-Rad, Hercules, CA, USA). For the Western blotting assay, 20 µg of protein mixed with 5 × SDS loading buffer was loaded per lane, separated with 10% SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk in TBST (TBS containing 0.05% Tween-20) for 1 h at room temperature. The membrane was then incubated at 4°C overnight with primary antibodies against TP53 (1:500, abcam), and GAPDH (1:1500, Santa Cruz Biotechnology, Inc.) followed by incubation with goat anti-mouse IgG (1:1000, Beijing Biosynthesis Biotechnology Co., Ltd.) or goat anti-rabbit antibody IgG (1:1000, Beijing Biosynthesis Biotechnology Co., Ltd.) for 1 h at room temperature. ECL Western blotting detection reagents (New England Biolabs, Ipswich, MA, USA) were used to visualize the target proteins, which were quantified with a Bio Image Intelligent Quantifier 1-D (Version 2.2.1, Nihon-BioImage Ltd., Japan).
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