For Western blot analysis, total proteins from the samples were separated by SDS-PAGE, transferred to nylon membranes and incubated separately with the following primary antibodies: MAPKs family kit/P-MAPKs family kit (dilution of 1: 1000; cat. no. 9926T/9910T, CST, Inc., MA, USA). B-actin (dilution of 1: 2000; cat. no. CW0096, CW BIO, Beijing, China) was used as an internal control. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody, goat anti-rabbit IgG (dilution, 1: 2000; cat. no. CW0103s; CW Bio, Beijing, China) or goat anti-mouse IgG (dilution, 1: 2000; cat. no. CW0102s; CW Bio, Beijing, China) at room temperature for 2 h, and the bands were visualized using chemiluminescence (Pierce Biotechnology, Inc., IL, USA). The images were analyzed using Fusion FX7 (Vilber Lourmat, Marne-la-Vallée, France).
Goat anti mouse igg
Manufactured by CWBIO
Sourced in China, United States
Goat anti-mouse IgG is a laboratory reagent used for the detection and quantification of mouse immunoglobulin G (IgG) in various immunoassays and research applications. It is produced by immunizing goats with purified mouse IgG and is designed to specifically bind to the Fc region of mouse IgG molecules.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by CWBIO (5 mentions)
Fusion fx7, by Vilber (3 mentions)
Chemiluminescence, by Thermo Fisher Scientific (2 mentions)
β actin, by CWBIO (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nlrp3, by Abcam (1 mentions)
Occludin, by Abcam (1 mentions)
Caspase 1, by Affinity Biosciences (1 mentions)
Ecl reagent, by CWBIO (1 mentions)
Mouse anti β actin mab, by CWBIO (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by CWBIO (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Gapdh, by GeneTex (1 mentions)
Gr68497 2, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
12 protocols using goat anti mouse igg
1
Western Blot Protein Analysis
2
Metformin Modulates Gut Inflammation
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Metformin (MET) was obtained from Sino-American Shanghai Squibb Pharmaceuticals, Ltd. (Shanghai, China). All assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mouse insulin (CSB-E05071m), LPS (CSB-E13066m), IL-18 (CSB-EO4609) and IL-1β (CSB-E08054) ELISA kits were provided by HuaMei Co. (Shanghai, China). Tissue lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (CWBIO) containing a protease/phosphatase inhibitor cocktail. The following primary antibodies were used: TLR4 (Affinity, San Francisco, CA, AF7017), NF-κB p65 (CST, Boston, MA, #8242), phospho-NF-κB p65 (CST, Boston, MA, #3033), NLRP3 (Abcam, Cambridge, UK, ab263899), ASC (Affinity, San Francisco, CA, AF6304), caspase-1 (Affinity, San Francisco, CA, AF5418), ZO-1 (CST, Boston, MA, #8193), occludin (Abcam, Cambridge, UK, ab216327) and β-actin (CWBIO, Beijing, China, CW0096). The secondary antibodies used were goat anti-rabbit IgG (CWBIO, Beijing, China) and goat anti-mouse IgG (CWBIO, Beijing, China).
3
PRRSV Infection and PARP Inhibition
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MARC-145 cells, plated in 12-well plates, were harvested after infection with PRRSV and treatment with 3-AB or DMSO at the indicated time points. Cell lysates were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by blotting onto polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). The membranes were primarily probed with rabbit anti-PARP antibody (Beyotime), swine anti-PRRSV-1 serum (Liu et al., 2010 ), or mouse anti-β-actin mAb (CWBIO, Beijing, PR China). After washing three times with TBS containing 0.1% Tween-20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (CWBIO), goat anti-swine IgG (Earthox, San Francisco, USA) or goat anti-mouse IgG (CWBIO) at 37 °C for 2 h, respectively. Detection was performed using enhanced chemiluminescence (ECL) reagents (CWBIO) according to the manufacturer's instructions.
4
Western Blot Analysis of Testicular Proteins
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Total protein was extracted from the frozen testicular tissues into RIPA buffer (Cat. No. CW2333S, CWBIO) that contained a phosphatase inhibitor cocktail (100×, Cat. No. CW2383S, CWBIO) and a protease inhibitor cocktail (100×, Cat. No. CW2200S, CWBIO). The protein concentration was quantified by the Pierce BCA Protein Assay Kit (Cat. No. 23227, Thermo Fisher Scientific, Waltham, MA, USA). Protein lysates (20 μg) were separated with 10% (w/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, IPVH00010, Millipore). Then, the membranes were blocked in a 5% (w/v) nonfat milk solution and incubated with antibodies for UCLH1 (dilution: 1:1000), CYP11A1 (dilution: 1:1000, Cat. No. GTX56293, Genetex, Irvine, CA, USA), SOX9 (dilution: 1:500), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; dilution: 1:4000, Cat. No. T0004, Affinity, Cincinnati, OH, USA) overnight at 4°C. Finally, the membrane was incubated with the appropriate secondary antibody (goat anti-rabbit IgG, 1:5000, CW0103S, CWBIO; goat anti-mouse IgG, 1:5000, CW0102S, CWBIO) and visualized with an enhancer (ChemiDoc Imaging System, BIO-RAD Laboratories, Hercules, CA, USA).
5
Protein Expression in Optical Tectum
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Animals were anesthetized and the tecta were dissected. The optical tecta (approximately 30–50 brains for each group) were homogenized in radioimmune precipitation assay (RIPA) buffer with a protease inhibitor cocktail (Sigma Aldrich) and phenylmethylsulfonyl fluoride (PMSF, Solarbio) at 4°C. Protein concentrations were measured by BCA assay using a Nanodrop (Thermo Scientific, 2000c). Protein homogenates were separated by SDS-PAGE (Bio-Rad). PVDF membranes were blocked in 4% non-fat milk for 1 h with TBS buffer containing 0.1% Tween-20 (Sigma Aldrich) (TBST) and incubated with primary antibodies overnight at 4°C. Antibodies were diluted in 1% non-fat milk. We used the following antibodies: anti-acetylation H3K9 (1:2000, Rabbit, Abcam, ab10812), anti-GAD67 (1:20000, Rabbit, Sigma, G5163), anti-VGAT (1:2000, Rabbit, ABclonal, A3129), and anti-GAPDH (1:10000, Rabbit, Millipore, GR68497-2). Blots were rinsed with TBST and incubated with the following horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature: goat anti-rabbit IgG (1:2000, CWbiotech, CW0103) and goat anti-mouse IgG (1:2000, CWbiotech, CW0102). Bands were visualized using ECL chemiluminescent (1:1, Pierce).
6
Protein Expression Analysis in Cell Samples
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The cells were collected and lysated with RIPA buffer (Byotime, Cat# P0013B) complemented with PMSF (Beyotime, Cat# ST506), protease inhibitor (Beyotime, Cat# P1050), and phosphatase inhibitors (Beyotime, Cat# P1096). Protein concentrations were determined using BCA protein assay kit (Thermo, Cat# 23225). SDS-PAGE electrophoresis was carried out in 4–20% precast polyacrylamide gels (Genscript, Nanjing, China, Cat# M00625) and blotted onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h with 5% skimmed milk powder in TBST and probed overnight at 4 °C with primary antibodies: phosphorylated-YAP (1:1000, Danvers, MA, USA, Cell Signaling, CAT#13008), YAP (1:2000, ABclonal, CAT#A1002), MYHC (1:1000, Abcam, CAT# ab37484), GAPDH (1:2000, Millipore, Darmstadt, Germany, CAT# MAB374). Secondary antibodies HRP conjugated goat anti-Rabbit IgG (Cwbiotech, Nanjing, China, Cat# CW0103S) or goat anti-mouse IgG (Cwbiotech, Cat# CW2333S) were diluted at 1:2000. Protein bands were visualized using SuperSignalTM West Pico Chemiluminescent Substrate (Thermo, Carlsbad, CA, USA, Cat# 34580) under ImageQuant 4000 (General Electric, Boston, NY, USA). For protein quantification, Quantity One was used to analyze grayscale values.
7
Molecular Pathways in Cancer Signaling
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SNS-032 (#S1145) and CDDP (#S1166) were purchased from Selleck Chemicals (Shanghai, China). The antibodies against RNA Pol II (#A300-653A), p-RNA Pol II (S5) (#A304-408A), and p-RNA Pol II (S2) (#A300-654A) were obtained from Bethyl Laboratories (Montgomery, TX). The anti-p-RNA Pol II (S7) (#04-1570) was purchased from EMD Millipore (Billerica, MA). The anti-CDK7 antibody (#2916), anti-CDK9 antibody (#2316), anti-Caspase-3 antibody (#9665), anti-PARP antibody (#9532), anti-Cleaved Caspase-3 antibody (#9664), anti-Bcl-XL antibody (#2762), anti-Bcl-w (31H4) (#2724), anti-XIAP antibody (#2042), anti-Mcl-1 antibody (#5453), anti-Cytochrome c antibody (#4272), anti-AIF antibody (#5318), anti-COX IV antibody (#4850), anti-MMP-2 antibody (#4022), and anti-GFP antibody (#2956) were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). The antibody against Ki67 (#ab15580) and MMP-1 (#ab137332) were obtained from Abcam (Cambridge, UK). The anti-Actin antibody (#4700) was purchased from Sigma (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies including Goat anti-Mouse IgG (#CW0102S) and Goat anti-Rabbit IgG (#CW0103S) were bought from CWBio (Beijing, China).
8
Protein Extraction and Western Blot Analysis
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Total protein extraction was carried out by lysing cells with 1× SDS loading buffer and denatured at 95°C for 5 min. The sample volume per lane was 10 μL. The protein samples were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, Billerica, USA). After being blocked and washed, the membranes were incubated overnight at 4°C with primary antibodies, followed by incubation with the corresponding secondary antibodies. Finally, the membrane was exposed with ECL developer (CW0049M; CWbio, Beijing, China) and processed with ImageJ software (
https://imagej.nih.gov/ij/ ). The primary antibodies used were as follows: anti-GAPDH (1:1000; CWbio), anti-HDAC6 (1:1000; 12834-1AP; Proteintech), anti-acetylated tubulin (1:1000; T7451; Sigma, St Louis, USA), anti-α-tubulin (1:1000; T6199; Sigma), anti-collagen II (1:1000; ab34712; Abcam), anti-MMP13 (1:1000; Wanleibio, Shenyang, China), and anti-SOX9 (1:1000; ab182579; Abcam). The secondary antibodies were goat anti-mouse IgG (1:4000; CWbio) and goat anti-rabbit IgG (1:4000; CWbio).
9
Purification and Western Blot Analysis of FoccCSP
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Following electrophoretic separation under denaturing conditions (12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)), purified FoccCSP was electroblotted onto a polyvinylidene difluoride (PVDF) membrane using a Bio-Rad Transblot transfer system (Bio-Rad Laboratories, Hercules, CA, USA). Following treatment with blocking solution (40 ml of PBS, 20 μl of Tween-20, 2 g of nonfat dried milk) overnight at 4°C, the membrane was incubated with anti-His tag mouse monoclonal antibody (Cwbio, Beijing, China) (1:2,000 dilution) for 1 h at 37°C, followed by goat anti-mouse IgG (Cwbio, Beijing, China) (1:2,500 dilution) for 2 h at 37°C. Finally, the protein band was visualized using a BCIP/NBT Alkaline Phosphatase Color Development Kit (Cwbio, Beijing, China).
10
Western Blot Analysis of Inflammasome Proteins
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In the western blotting analysis, the samples were incubated with the inflammasome antibody sampler kit (cat. no. 32961T, CST Inc., MA, USA), anti-human gasdermin D (cat. no. 96458S, CST Inc., MA, USA), anti-rat IL-1β (cat. no. 500-P80-100, PeproTech, NJ, USA), anti-rat IL-18 (cat. no. ab191860, Abcam, UK), anti-rat caspase-1 (cat. no. ab1872, Abcam, UK), anti-rat collagen II (cat. no. ab34712, Abcam, UK), and anti-rat aggrecan (cat. no. ab36861, Abcam, UK) overnight, then incubated with horseradish peroxidase-conjugated secondary antibody, goat anti-rabbit IgG (1 : 2,000 dilution; cat. no. CW0103s; CW Bio, Beijing, China), or goat anti-mouse IgG (1 : 2,000 dilution; cat. no. CW0102s; CW Bio, Beijing, China) at room temperature for 2 h. GAPDH and β-actin were used as the internal controls. The bands were visualized using chemiluminescence (Pierce Biotechnology Inc., IL, USA) and analyzed using Fusion FX7 (Vilber Lourmat, Marne-la-Vallée, France).
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