Cultured cells were treated with lysis buffer and mechanically degraded to release the proteins. Protein concentration was determined using modified Lowry method [25 (link)]. Loading buffer (0.125 M Tris-HCl, pH 6.8, 2% SDS, 0.5% (v/v) 2-mercaptoethanol, 1% bromophenolblue and 19% glycerol) was added to protein sample and heated for 5 min at 95ºC. Proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membranes in a humid environment using a transfer buffer (25mM Tris, 190mM glycine, 20% methanol). Membranes were blocked with 5% milk in TBS-T (0.05% Tween-20) and incubated with primary antibodies overnight at 4ºC. Membranes were washed 3 times with wash buffer TBS-T and incubated with a secondary anti-rabbit IgG or anti-mouse IgG (Cell Signalling Technologies Danvers, MA) antibody conjugated to the enzyme horseradish peroxidase (HRP) for 1 h. Membranes were washed three times and proteins were detected using the ECL method as specified by the manufacturer. Autoradiography signals were assessed using digital image system ImageQuant LAS 4000 (GE Healthcare).
Anti rabbit igg or anti mouse igg
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-rabbit IgG or anti-mouse IgG are secondary antibodies used to detect the presence of rabbit or mouse primary antibodies in various immunoassays, such as Western blotting, ELISA, and immunohistochemistry. These secondary antibodies are conjugated with reporter molecules, such as enzymes or fluorescent dyes, to enable the visualization and quantification of the target primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000, by GE Healthcare (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Timp 1, by Cell Signaling Technology (1 mentions)
Timp 2, by Cell Signaling Technology (1 mentions)
Hrp linked antibody, by Cell Signaling Technology (1 mentions)
Caspase 12, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Ire1α, by Cell Signaling Technology (1 mentions)
Cdc42, by Cell Signaling Technology (1 mentions)
P eif2α, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Caspase 3, by Beyotime (1 mentions)
Bicinchoninic acid protein assay, by Beyotime (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Ecl western blotting substrate, by Merck Group (1 mentions)
Sds page sample loading buffer, by Beyotime (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
8 protocols using anti rabbit igg or anti mouse igg
1
Western Blot Protein Detection Protocol
2
Evaluating Cellular Stress Responses to GE
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HEY and A2780 cells were seeded into 6-well plates and treated with GE. After the treatment was administered, the cells were lysed in RIPA lysis buffer (Beyotime) in an ice bath for 20 min. Protein concentration was determined by utilizing a BCA protein assay kit. Total proteins (20 μg) were subjected to 8–12% SDS-PAGE, transferred onto polyvinylidene fluoride membranes, and blocked with 5% nonfat milk in PBS with Tween 20 (PBST) at room temperature for 1 h. The membranes were probed with specific primary antibodies against p-eIF2α, IRE-1α, BiP, CHOP, caspase-12, PARP, Bcl-xL, GAPDH, RhoA, Rac, Cdc42, MMP-9, MMP-2, TIMP-1, and TIMP-2 (Cell Signaling Technology, Beverly, MA, USA), and caspase-3 (Beyotime) overnight at 4 °C. The membranes were washed with PBST thrice and then incubated with anti-rabbit IgG or anti-mouse IgG with a HRP-linked antibody (Cell Signaling Technology). Equal protein loading was verified by probing with GAPDH antibody. Images were obtained using a ChemiDoc MP Imaging System, and quantification was performed with Image Lab 5.1. The experiments in this assay were performed at least three times.
3
Western Blot Protein Analysis Protocol
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Cultured cells were treated with lysis buffer and then mechanically degraded to release the proteins. Protein concentration was determined using modified Lowry method 16 . Loading buffer (0.125 M Tris-HCl, pH 6.8, 2% SDS, 0.5% (v/v) 2-mercaptoethanol, 1% bromophenolblue and 19% glycerol) was added to protein sample and heated for 5 min at 95ºC. Proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membranes in a humid environment using a transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol). Membranes were blocked with 5% milk in TBS (0.05% Tween-20) and were incubated with primary antibodies overnight at 4ºC. Membranes were washed 3 times with wash buffer TBS-T (TBS, 0.2% Tween-20) and were incubated with a secondary anti-rabbit IgG or anti-mouse IgG (Cell Signalling Technologies Danvers, MA) antibody conjugated to the enzyme horseradish peroxidase (HRP) for 1 h. Membranes were washed three times and proteins were detected using the ECL method as specified by the manufacturer. Autoradiography signals were assessed using digital image system ImageQuant LAS 4000 (GE Healthcare).
4
Protein Extraction and Western Blot Analysis
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Cells were dissolved in RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with proteinase inhibitor cocktail and phosphatase inhibitor cocktail (Sigma, United States) for 15 min. Protein concentrations were determined by the bicinchoninic acid protein assay (Beyotime, China). Equal amounts of protein were solubilized in SDS-PAGE Sample Loading Buffer (Beyotime, China), boiled for 5 min, and then separated by SDS-PAGE. After transfer, the membranes were blocked with TBST buffer containing 5% nonfat milk, followed by incubation with primary antibodies overnight at 4 °C. After incubation, the membranes were incubated with the following anti-rabbit IgG or anti-mouse IgG (Cell Signaling Technology, United States) at room temperature for 1 h. The bands were visualized by ECL western blotting substrate (Millipore, MA, United States).
5
Mammary Gland Protein Extraction and Western Blot
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Mammary gland tissues were collected at 15 weeks of age and snap-frozen in liquid nitrogen prior to lysate preparation. The tissues were homogenized at 4°C in NP40-based cell lysis buffer (50 mM Tris•HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and centrifuged at 18,000 × g for 20 min at 4°C. The proteins in the supernatant were collected and quantified using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Total protein (50 µg) from each sample was separated by 10–12% SDS-PAGE and transferred onto nitrocellulose membranes. Following blocking in 5% non-fat milk in Tris-buffered saline/Tween-20 (TBST) for 1 h at room temperature, membranes were incubated with the indicated primary antibodies (Table I ) overnight at 4°C, followed by washing with TBST. After incubation with horseradish peroxidase-linked secondary antibodies (1:5,000 dilution; anti-mouse IgG or anti-rabbit IgG; Cat. #7076 or #7074; Cell Signaling Technology) for 1 h at room temperature and washing with TBST, specific protein bands were visualized with SuperSignal West Pico Chemiluminescent solution (Thermo Fisher Scientific, Inc.). Protein bands were imaged using the FluorChemE imaging system.
6
Immunohistochemical Analysis of Placental Tissue
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Human placental tissues were collected in accordance with the policy of the Ethics Committee of the First Affiliated Hospital of Jinan University. These tissues were fixed in 4% paraformaldehyde for 24 h following a standard protocol, then dehydrated and embedded in paraffin. Next, paraffin sections were sliced on microtome at a thickness of 5 μm. Mouse anti-NQO1 antibodies (1:200, Cat. ab28947, Abcam) and rabbit anti-SRXN1 (1:100, Affinity Biosciences Cat# DF12028, RRID:AB_2844833) were used as primary antibodies, and anti-mouse IgG or anti-rabbit IgG was used as a secondary antibody (1:1,000, Cell Signaling Technology, United States). The reaction was revealed using Novolink Polymer (Leica Mycrosystems, Newcastle upon Tyne, United Kingdom) followed by diaminobenzidine (DAB, Dako, Carpinteria, CA, United States) as a chromogen. The sections were then counterstained with Mayer’s hematoxylin. The results of the statistical analysis of immunohistochemistry were expressed by relative optical density (IOD).
7
Western Blot Analysis of Inflammasome Markers
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Samples and markers were applied to gels, which were run at 120 V. Subsequently, gels were blotted using the Trans-Blot Turbo Transfer System (BioRad, Berkely, CA, USA). Membranes were blocked for 1 h and primary antibodies ASC (B-3) (Santa Cruz Biotechnology, INC., Dallas, TX, USA; 1:500 in 5% dried milk), NLRP3 (Cell Signaling Technology, Danvers, MA, USA; 1:1000 in 5% BSA in TBST buffer), P-NFkB (Cell Signaling Technology; 1:1000 in 5% dried milk), cleaved Interleukin-1β (Cell Signaling Technology; 1:1000 in 5% dried milk), IL-1β (Cell Signaling Technology; 1:1000 in 5% BSA in TBST buffer), cleaved GSDMD (Cell Signaling Technology; 1:500 in 5% dried milk) or GSDMD (Merck; 1:500 in 5% dried milk) were added and incubated at 4 °C overnight. After washing, anti-mouse IgG or anti-rabbit IgG (both Cell Signaling Technology; 1:5000 in 5% dried milk) were added for 1 h. Membranes were developed using the Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and visualized using the FUSION SOLO S imaging system (Vilber Lourmat, Eberhardzell, Germany).
8
Kidney Protein Expression Analysis
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Kidney samples (20 mg) were homogenized in RIPA lysis buffer containing complete protease inhibitor cocktail (Roche). Protein concentration was determined by the BCA Assay. Samples were mixed with 2x Laemmli buffer and boiled. Equal amounts of protein (40 µg) were separated on 10% SDS-polyacrylamide gel, transferred to nitrocellulose membranes and blocked with 5% skim milk in Tris-buffered saline (TBS), containing 0.1% Tween-20. Membranes were incubated overnight at 4 °C with primary antibodies (rabbit polyclonal anti-TGF-β1 antibody 1:500, SantaCruz Biotechnology, Santa Cruz, CA, USA; rabbit polyclonal anti-PKG 1:2000, Enzo, Farmingdale, NY, USA; rabbit polyclonal anti-sGCß1 1:2000, Novus Biologicals; rabbit polyclonal anti-PDE5a 1:1000, Enzo; p44/42 MAPK (ERK1/2) and phospho-p44/42 MAPK (pERK1/2) (Thr202/Tyr204) both at 1:1000, Cell Signaling, Danvers, MA, USA), then washed and incubated with peroxidase-conjugated secondary antibody (anti-mouse IgG or anti-rabbit IgG, 1:2000, Cell Signaling). Blots were visualized by ECL detection kit (Pierce Thermo). Original immunoblots are shown in Supplementary Figure 2 for PDE-5, TGF-ß1 and p-ERK and in Supplementary Figure 3 for PKG and sGCß1.
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