The spinal cord tissues (L3-L4) were homogenized in a lysis buffer containing Protease Inhibitor Cocktail (p8340, Sigma-Aldrich) [19 (link)]. The protein concentration was confirmed with BCA protein concentration determination assay kit (P0010, Beyotime, Shanghai, China). The protein samples were separated by SDS-PAGE gels and transferred onto polyvinylidene fluoride membrane (IPVH00010, Millipore). The membrane was sealed with 5% skim milk for 2 h at room temperature, subsequently incubated at 4°C overnight with the primary antibodies: Mtf1 (1 : 500; Santa Cruz Biotechnology, CA, USA), p-ERK1/2 (1 : 5000; Sigma), ERK1/2 (1 : 1000; Santa Cruz Biotechnology), GFAP (1 : 1000; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1 : 2000; Santa Cruz Biotechnology). The membrane was washed for 5 min/time 3 times at room temperature, incubated for 2 h in the corresponding secondary antibody: HRP-labeled goat anti-mouse IgG (1 : 1000; Beyotime), HRP-labeled goat anti-rabbit IgG (1 : 1000; Beyotime) and HRP-labeled donkey anti-goat IgG (1 : 1000; Beyotime) at room temperature. The membrane was then washed for 5 min/time 6 times, the immune complexes were detected by ECL chemiluminescent assay kit (Biosharp, Guangzhou, China). Signal intensity of band analyses was conducted with ImageJ software (Alliance Q9).
Hrp labeled goat anti mouse igg
Manufactured by Beyotime
Sourced in China, United States
HRP-labeled goat anti-mouse IgG is a secondary antibody conjugated with Horseradish Peroxidase (HRP). It is used to detect and quantify mouse immunoglobulin G (IgG) in various immunoassays and immunohistochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Hrp labeled goat anti rabbit igg, by Beyotime (30 mentions)
Hrp labeled donkey anti goat igg, by Beyotime (4 mentions)
Horseradish peroxidase hrp labeled goat anti rabbit igg, by Beyotime (4 mentions)
Microplate reader, by Agilent Technologies (4 mentions)
Tmb chromogen solution, by Beyotime (4 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Surepage gel, by GenScript (3 mentions)
A0216, by Beyotime (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Dylight 488 goat anti mouse igg h l 70 gam4882, by MultiSciences Biotech (3 mentions)
Dylight 594 goat anti rabbit igg h l 70 gar5942, by MultiSciences Biotech (3 mentions)
Goat anti rabbit igg, by Beyotime (3 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescent reagent, by Beyotime (2 mentions)
Anti β actin, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Aa128, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
G box system, by Syngene (2 mentions)
55 protocols using hrp labeled goat anti mouse igg
1
Western Blot Analysis of Spinal Cord Proteins
2
Western Blot Analysis of Protein Expression
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Samples were treated with RIPA Lysis Buffer (Solarbio, Beijing, China) to extract the total protein. The proteins were quantified and stored at −20°C before use. We prepared 10% sodium dodecyl sulfate polyacrylamide gel to isolate the proteins. After transfer to nitrocellulose membrane, the bands were blocked with 5% non-fat milk. Then, the corresponding primary antibodies and secondary antibodies were diluted to appropriate concentrations and added to the protein bands, respectively. Finally, the protein bands were scanned with Tanon 5200 (Tanon, Shanghai, China). Integrated density value was used to calculate the relative protein quantity. The antibodies and reagents used were: ARHGAP24 (Abcam, ab203874, 1: 500); MMP9 (Abcam, ab76003, 1: 1000); VEGF (Abcam, ab69479, 1: 1000); Vimentin (Cell Signaling Technology, #5471, 1: 1000); E-cadherin (Cell Signaling Technology, #14472, 1: 1000); β-catenin (Abcam, ab32572, 1: 5000); GAPDH (Cell Signaling Technology, #5174, 1: 2000); HRP-labeled Goat Anti-Rabbit IgG (Beyotime, A0208, 1: 1000); HRP-labeled Donkey Anti-Goat IgG (Beyotime, A0181, 1: 1000); HRP-labeled Goat Anti-Mouse IgG (Beyotime, A0216, 1: 1000).
3
Protein Extraction from Transfected Eel Liver Cells
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Japanese eel liver cells were used, and the proteins were extracted 24 h after transfection. The overexpressed cells were washed twice with PBS, then lysed by adding Western and IP cell lysates (RIPA, Beyotime, Shanghai, China) for 15 min on ice, and centrifuged at 12,000× g for 5 min, and the supernatant containing the proteins was retained. Then, 2× SDS Protein Sampling Buffer was added to the supernatant, boiled for 15 min, and stored at −80 °C for Western blot analysis. His Tag Mouse Monoclonal Antibody (Product No. AF5060, Beyotime, Shanghai, China) was used as the primary antibody and HRP-labeled Goat Anti-Mouse IgG (Product No. A0216, Beyotime, Shanghai, China) was used as the secondary antibody.
4
Western Blot Analysis of Protein Targets
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Equal amounts of protein (20–30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF, Millipore, Billerica, MA, USA). The membrane was blocked for 1 h with 5% fat-free dried milk at RT, then incubated with primary antibodies (Santa Cruz Biotechnology) against A3AR (1:800 dilution), NF-κB p65 (1:600 dilution), IκB-α (1:400 dilution), p-IκB-α (1:400 dilution), Tubulin (1:1,000 dilution), or β-actin (1:1,000 dilution) at 4°C overnight. Membranes were washed three times with Tris-buffered saline with Tween-20 (TBS-T) and incubated with the corresponding secondary antibodies (HRP-labeled Goat Anti-mouse IgG, HRP-labeled Goat Anti-rabbit IgG, HRP-labeled Donkey Anti-goat IgG; 1:1,000; Beyotime, China) for 1 h at room temperature. Signals were detected with an electrochemiluminescence (ECL) detection reagent (Beyotime, China). The images were obtained on Kodak film and quantified by Quantity One software (Bio-Rad, Hercules, CA, USA).
5
Gastric Cancer Drug Resistance Model
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The human gastric cancer cell line BGC823 was purchased from KeyGEN BioTECH Co., Ltd. (China).
The drug-resistant cell line BGC823/5-Fu was established by the low-dose multiple shock method in our previous research [17 (link)] and stored at -196 °C. The drug resistance index of BGC823/5-Fu was 13.
The serum-free medium (SFM) was composed of 95% DMEM/F12 (Gibco, USA), 1% 2 µg/ml EGF (Peprotech, USA), 1% 2 µg/ml bFGF (Peprotech, USA), 2% B27 (Gibco, USA), 1% 100 U/ml penicillin/streptomycin (Gibco, USA) and 0.4 U insulin (Sigma, USA). The IGF-1 was purchased from Sigma (USA). The P-gp, MRP1, PI3K, p-PI3K, AKT, p-AKT, Nrf2 and β-actin antibodies were purchased from Bioss, Inc. (China) and Proteintech Group, Inc. (USA). HRP-labeled Goat Anti-Rabbit IgG, HRP-labeled Goat Anti-Mouse IgG and Alexa Fluor 488-labeled Goat Anti-Rabbit IgG were purchased from Beyotime Biotechnology (China). The Annexin V-FITC/PI Kit was purchased from KeyGEN BioTECH Co., Ltd. (China). CD44 MicroBead Kit was purchased from Miltenyi Biontec. (Germany). Lipofectamine2000 was purchased from Invitrogen (USA).
The drug-resistant cell line BGC823/5-Fu was established by the low-dose multiple shock method in our previous research [17 (link)] and stored at -196 °C. The drug resistance index of BGC823/5-Fu was 13.
The serum-free medium (SFM) was composed of 95% DMEM/F12 (Gibco, USA), 1% 2 µg/ml EGF (Peprotech, USA), 1% 2 µg/ml bFGF (Peprotech, USA), 2% B27 (Gibco, USA), 1% 100 U/ml penicillin/streptomycin (Gibco, USA) and 0.4 U insulin (Sigma, USA). The IGF-1 was purchased from Sigma (USA). The P-gp, MRP1, PI3K, p-PI3K, AKT, p-AKT, Nrf2 and β-actin antibodies were purchased from Bioss, Inc. (China) and Proteintech Group, Inc. (USA). HRP-labeled Goat Anti-Rabbit IgG, HRP-labeled Goat Anti-Mouse IgG and Alexa Fluor 488-labeled Goat Anti-Rabbit IgG were purchased from Beyotime Biotechnology (China). The Annexin V-FITC/PI Kit was purchased from KeyGEN BioTECH Co., Ltd. (China). CD44 MicroBead Kit was purchased from Miltenyi Biontec. (Germany). Lipofectamine2000 was purchased from Invitrogen (USA).
6
Western Blotting for Protein Analysis
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Western blotting was performed as previously described (Gong et al., 2013 (link)). The primary antibodies used were as follows: anti-p38 MAPK (Cell Signaling Technology, 1:1000); anti-phospho-p38 MAPK (Cell Signaling Technology, 1:1000); anti-GAPDH (Cell Signaling Technology, 1: 1000); anti-Bcl-2 (Affinity Biosciences, 1: 1000); anti-Bax (Cell Signaling Technology, 1: 1000); anti-β-actin (Cell Signaling Technology, 1: 1000); anti-p53 (Cell Signaling Technology, 1: 1000); anti-phosphor-p53 (Affinity Biosciences, 1: 1000); HRP-labeled goat anti-rabbit IgG (Beyotime Biotechnology, 1: 1000), and HRP-labeled goat anti-mouse IgG (Beyotime Biotechnology, 1: 1000). All experiments were performed at least three times (i.e., three separate protein preparations) under the same conditions.
7
Western Blot Analysis of Protein Targets
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The total cellular protein was extracted with RIPA strong lysate. The protein samples were electrophoresed on 10% or 15% polyacrylamide gel and transferred to PVDF membranes, which were blocked with a 5% solution of non-fat milk at room temperature for 1.5 h. GAPDH antibody (Proteintech, 66009-1-Ig, 1:10,000), IFIT3 antibody (Santa Cruz, sc-393512, 1:500), IFIT5 antibody (Proteintech, 13378-1-ap, 1:1000), and PRRSV N antibody (BIOSs, bs-23941r, 1:1000) were added, respectively, and incubated overnight at 4 °C. After washing the PVDF membrane with TBST 4 times, HRP-labeled Goat anti-mouse IgG (Beyotime, a0216) or HRP-labeled Goat anti-rabbit IgG (Beyotime, a0208) solutions were added and incubated at room temperature for 1.5 h. According to the manufacturer’s instructions, the protein bands were visualized on the chemiluminescence imager (Shanghai Tianneng Technology Co., Ltd., Tanon-5200) using an ECL kit (Vazyme, E412), and the gray value of the protein bands was analyzed with ImageJ software.
8
Protein Isolation and Western Blot Analysis
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The total protein of ovary tissues was isolated by the proteinase inhibitor-containing lysis buffer. The 4% SurePAGE gel (GenScript, Nanjing, China) was used to separate the equivalent amounts of protein. The separated proteins were placed onto a PVDF membrane (Pall, Mexico) after electrophoresis, and then blocked with sealing solution (Tiangen, Beijing, China). The blocked membrane was incubated overnight at 4°C with anti-JAK3 (1:500; Santa Cruz, Cambridge, UK), anti-CDK4 (1:1,000), anti-cyclin D2 (1:1,000) (all from Cell Signaling Technology, US), and GAPDH (1:2,000; Proteintech, Chicago, IL, USA). After rinsing 3 times with Tris-buffered saline/Tween, the corresponding HRP-labeled Goat Anti-Rabbit IgG (1:1,000; Beyotime) or HRP-labeled Goat Anti-Mouse IgG (1:1,000; Beyotime) was used to incubate the membranes for 1 h at room temperature. The Enhanced Chemiluminescent Reagent (Beyotime) was used to visualize the protein blots.
9
Western Blot Detection of His-tagged Protein
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PTD-CcTrx1 was identified by immunoblotting. After electrophoresis on 12% SDS polyacrylamide gel, proteins were transferred to PVDF membrane and blocked with Western blot blocking liquid (Beyotime, Shanghai, China) for 1 h at room temperature. Transferred proteins were treated with the anti-His-tag antibody from mice (1:2000 dilution, Tiangen, Beijing, China) overnight at 4 °C, and then incubated with the HRP-labeled goat anti-mouse IgG (Beyotime, Shanghai, China) for 1 h at room temperature. Afterward, the membranes were washed for 30 min and then visualized using a chemiluminescent detection system (Syngene G: Box, Cambridge, UK).
10
Optimized Antibody Panel for Immunoblotting and Flow Cytometry
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The following antibodies were used for immunoblotting: Cell Signaling Technology, anti-p38 (Cat#: 8690), anti-p-p38 (Cat#: 4511), anti-p65 (Cat#: 8242), anti-p-p65 (Cat#: 3033), anti-JNK (Cat#: 9252), anti-p-JNK (Cat#: 4668), anti-Erk (Cat#: 4695), anti-p-Erk (Cat#: 4370), anti-PKM2 (Cat#: 4053), anti-STAT3 (Cat#: 12640); Abcam, anti-Pyk2 (Cat#: 228477); Santa Cruz Biotechnology, anti-p-Pyk2 (Cat#: sc-81512); Beyotime Institute of Biotechnology, anti-β-actin (Cat#: AA128), HRP labeled Goat Anti-Rabbit IgG (Cat#: A0208), HRP-labeled Goat Anti-Mouse IgG (Cat#: A0216). The following antibodies purchased from Biolegend were used for flow cytometry: FITC anti-mouse B220 (Cat#: 103206), BV421 anti-mouse CD11c (Cat#: 117330), FITC anti-mouse F4/80 (Cat#: 123108), APC anti-mouse CD86 (Cat#: 105012), PE anti-mouse CD40 (Cat#: 124610), PE anti-mouse GL7 (Cat#: 144607), APC anti-mouse CD95 (Cat#: 152604), APC anti-mouse CXCR5 (Cat#: 145506), PE anti-mouse PD-1 (Cat#: 135206), APC/Cy7 anti-human CD19 (Cat#: 302218), FITC anti-human CD14 (Cat#: 367116), BV421 anti-human CD11c (Cat#: 301628), APC anti-human CD86 (Cat#: 374208), PE anti-human CD40 (Cat#: 334308). All the antibodies for flow cytometry were used at a 1:100 dilution.
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