MARC-145 cells seeded in six-well plates were infected with PRRSV. At 24 hpi, the cells were harvested with 1 × SDS loading buffer (Beyotime) and boiled for 10 min. After centrifuging at 12,000× g, 4 °C for 10 min, proteins were separated by electrophoresis on 12% polyacrylamide gels (EpiZyme Biotechnology, Shanghai, China) and electro-blotted onto polyvinylidene difluoride membranes (Millipore, MA, USA). Next, the membranes were blocked at room temperature with 5% BSA for 3 h and incubated with the antibody specific to PRRSV-N protein, followed by incubation with horseradish peroxidase-conjugated Goat Anti-Mouse IgG (Beyotime). Finally, the resultant images were visualized and collected using Clarity Western Peroxide Reagent (Bio-Rad, CA, USA).
Horseradish peroxidase conjugated goat anti mouse igg
Manufactured by Beyotime
Sourced in China
Horseradish peroxidase-conjugated goat anti-mouse IgG is a secondary antibody conjugated with the enzyme horseradish peroxidase. It is designed to detect and bind to mouse immunoglobulin G (IgG) in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Sds loading buffer, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Clarity western peroxide reagent, by Bio-Rad (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti gp64 antibody, by Abcam (1 mentions)
Loading buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Chemiluminescence imaging system, by Clinx (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Ecl plus western blotting detection kit, by GE Healthcare (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Ecl western blotting detection system, by Bio-Rad (1 mentions)
8 protocols using horseradish peroxidase conjugated goat anti mouse igg
1
Immunoblotting Analysis of PRRSV-N Protein
2
Western Blot Analysis of Protein Expression
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Cells were washed twice with PBS and lysed with 50 μl protein lysis buffer (Beyotime, Shanghai, China) containing 2% PMSF (Beyotime). After 30 min on ice, lysates were treated with 20% loading buffer (Beyotime) for 10 min at 100°C. Proteins were resolved by 15% SDS-PAGE and analyzed by immunoblotting using anti-Flag antibody (1:2000, Sigma, Burghausen, Germany) and anti-GP64 antibody (1:2000, Abcam, Cambridge, USA). The horseradish peroxidase-conjugated goat anti-mouse IgG (1:20000, Beyotime) was used as the secondary antibody. Protein bands were visualized using the Supersignal Western Femto Maximum Sensitivity Substrate Western Blotting Kit (Thermo, Waltham, MA, USA) and a Chemiluminescence Imaging System (Clinx Science Instruments, Shanghai, China).
3
Western Blot Analysis of BmCYP18A1 Protein
4
Western Blot Analysis of Apoptosis and EMT
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Protein samples were preprocessed with RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.). The protein (30 µg/lane) was separated by 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). The membranes were blocked with 10% non-fat milk in 1X TBS + 0.1% Tween-20 (TBST) for 2 h at room temperature. Subsequently, the membranes were incubated with primary antibodies against cleaved caspase-3 (1:1,000; ab2302), Bax (1:1,000; ab32503), Bcl-2 (1:1,000; ab32124), E-cadherin (1:50; ab1416), vimentin (1:1,000; ab92547), Snail (1:1,000; ab229701), AKT (1:10,000; ab179463), phosphor (p)-AKT (T308; 1:1,000; ab38449), mTOR (1:2,000; ab2732), p-mTOR (1:1,000; ab109268) and anti-GAPDH (1:10,000; ab181602) at 4°C overnight. All primary antibodies were obtained from Abcam. Subsequently, the membranes were washed three times with TBST and incubated with a horse-radish peroxidase-conjugated goat-anti-mouse IgG (1:1,000; A7007; Beyotime Institute of Biotechnology) for 1 h at room temperature. Specific protein bands were developed using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) and visualized using a ChemiDoc MP system (Bio-Rad Laboratories, Inc.).
5
Western Blot Analysis Protocol
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Cells were washed three times with PBS and lysed in cell lysis buffer (Beyotime P0013). The cell lysate was mixed with 5× SDS loading buffer (Beyotime P0015), resolved with 12% acrylamide–SDS-PAGE buffer, and electroblotted onto polyvinylidene difluoride membranes. The membranes were blocked with 10% nonfat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBST) for 1 h and incubated with the indicated antibodies at room temperature for 3 h. Following three washes with TBST, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Beyotime A0216) or goat anti-rabbit IgG (Beyotime A0208) at room temperature for 1 h. Signals were visualized using a ChemiDoc Touch imaging system from Bio-Rad and quantitated using ImageJ software.
6
Western Blotting of Tagged Proteins
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Collected cell samples and silkworm samples were processed by lysate, and after SDS-PAGE, the proteins were transferred into PVDF membranes (Roche Diagnostics, Basel, Switzerland) and incubated with the primary antibody Flag (Beyotime, Suzhou, China), and the secondary antibody, horseradish peroxidase-conjugated goat anti-mouse IgG (Beyotime). The results of Western blotting were analyzed by a ECL Western blotting Detection System (Bio-Rad, Hercules, CA, USA).
7
Western Blot Analysis of rSjVAMP2 Protein
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At 6 days post-siRNA treatment, soluble proteins were extracted from worms and quantified by using the Bradford protein assay (Sangon Biotech, Shanghai, China). Subsequently, the proteins were separated by SDS-PAGE, electrotransferred onto a PVDF membrane (Whatman International Ltd., Kent, UK), and blocked in 5% skim milk in PBS (pH 7.4) containing 0.1% Tween 20 (Sigma, St Louis, MO, USA) (PBST) overnight at 4 °C. The membrane was incubated with specific mouse anti-rSjVAMP2 serum43 (link) at a dilution of 1:100 or β-actin (Cell Signaling Technology, Boston, MA, USA) diluted 1:1000 in PBST for 1 h at room temperature. After three 5-min washes in PBST, the bound primary antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse IgG (Beyotime Biotechnology, Shanghai, China) diluted 1:2000 for 1 h at room temperature. The membrane-bound proteins were exposed to an X-ray film and detected using the enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Stockholm, Sweden).
8
Stem Cell Factor and Cytokine Stimulation
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Recombinant human stem cell factor (SCF), IL-1β, IL-3, and IL-6 were purchased from Peprotech (Rocky Hill, NJ). Serum-free methylcellulose medium (SF H4236) was purchased from StemCell Technologies (Vancouver, Canada). Dulbecco's phosphate-buffered saline (PBS), Iscove's modi ed Dulbecco's medium (IMDM), RPMI-1640, fetal calf serum (FCS) and bovine serum albuminwere purchased from Gibco BRL (NY, USA). Rabbit anti-human E-cadherin,mouse anti-human vimentin monoclonal antibody (mAb) from Boster (Wuhan, China). Mouse anti-TGF-β1 mAb was from R&D Systems (Minneapolis, MN). Horseradish peroxidase-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-mouse IgG were purchased from Beyotime Biotechnology (Shanghai, China).
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